Pharmaceutical composition comprising a bispecific antibody for epcam

ABSTRACT

The present invention provides a pharmaceutical composition comprising a bispecific single chain antibody construct. Said bispecific single chain antibody construct is characterized to comprise or consist of at least two domains, whereby one of said at least two domains specifically binds to human EpCAM and comprises at least one CDR-H3 region comprising the amino acid sequence NXID antigen and a second domain binds to human CD3 antigen. The invention further provides a process for the production of the pharmaceutical composition of the invention, a method for the prevention, treatment or amelioration of a tumorous disease and the use of the disclosed bispecific single chain antibody construct and corresponding means in the prevention, treatment or amelioration of a tumorous disease.

The invention relates to a pharmaceutical composition comprising abispecific single chain antibody construct. Said bispecific single chainantibody construct is characterized to comprise or consist of at leasttwo domains, whereby one of said at least two domains specifically bindsto human EpCAM antigen and comprises at least one CDR-H3 regioncomprising the amino acid sequence NXD and a second domain binds tohuman CD3 antigen. The invention further provides a process for theproduction of the pharmaceutical composition of the invention, a methodfor the prevention, treatment or amelioration of a tumorous disease andthe use of the discloSed bispecific single chain antibody construct andcorresponding means in the prevention, treatment or amelioration of atumorous disease.

A variety of documents is cited throughout this specification. Thedisclosure content of said documents is herewith incorporated byreference.

Epithelial cell adhesion molecule (EpCAM, also called 17-1A antigen,KSA, EGP40, GA733-2, ks1-4 or esa) is a 40-kDa membrane-integratedglycoprotein of 314 amino acids with specific expression in certainepithelia and on many human carcinomas (reviewed in Baizar, J. Mol. Med.1999, 77, 699-712). EpCAM was discovered and subsequently cloned throughits recognition by the murine monoclonal antibody 17-1A/edrecolomab(Goettlinger, Int J Cancer. 1986; 38, 47-53 and Simon, Proc. Natl. Acad.Sci. USA. 1990; 87, 2755-2759). Monoclonal antibody 17-1A was generatedby immunization of mice with human colon carcinoma cells (Koprowski,Somatic Cell Genet. 1979, 5, 957-971).

The EGF-like repeats of EpCAM were shown to mediate lateral andreciprocal interactions in homophilic cell adhesion (Balzar, Mol. Cell.Biol. 2001, 21, 2570-2580) and, for that reason, is predominantlylocated between epithelial cells (Litvinov, J Cell Biol. 1997, 139,1337-1348, Balzar, J Mol Med. 1999, 77, 699-712 and Trebak, J Biol Chem.2001, 276, 2299-2309). EpCAM serves to adhere epithelial cells in anoriented and highly ordered fashion (Litvinov, J Cell Biol. 1997, 139,1337-1348). Data from experiments with transgenic mice and ratsexpressing human EpCAM on their epithelia suggest that EpCAM on normaltissue may however not be accessible to systemically administeredantibody (McLaughlin, Cancer Immunol. Immunother., 1999, 48, 303-311).Upon malignant transformation of epithelial cells the rapidly growingtumor cells are abandoning the high cellular order of epithelia.Consequently, the surface distribution of EpCAM becomes less restrictedand the molecule better exposed on tumor cells. Due to their epithelialcell origin, tumor cells from most carcinomas still express EpCAM ontheir surface.

In vivo, expression of EpCAM is related to increased epithelialproliferation and negatively correlates with cell differentiation (forreview see Balzar, 1999, J. Mol. Med. 77, 699-712). Expression of EpCAM,as detected by immunohistochemistry using anti-EpCAM monoclonalantibodies, is essentially seen with all major carcinomas (reviewed inBalzar, J Mol Med. 1999, 77, 699-712). Best EpCAM expression wasobserved with non-small cell lung cancer (De Bree, Nucl Med Commun.1994, 15, 613-27) and prostate cancer (Zhang, Clin Cancer Res. 1998, 4,295-302) where 100% of tumor patient samples showed positive EpCAMstaining. In these studies, EpCAM is also reported to homogeneouslystained tumor tissues indicating that the antigen is expressed on alarge proportion of cells of a′ given tumor. Because of its widespreadexpression, EpCAM is referred to as a “pan-carcinoma” antigen.

EpCAM has been shown in various studies to be beneficial in diagnosisand therapy of various carcinomas. Furthermore, in many cases, tumorcells were observed to express EpCAM to a much higher degree than theirparental epithelium or less aggressive forms of said cancers. Forexample, EpCAM expression was shown to be significantly higher onneoplastic tissue and in adenocarcinoma than on normal prostateepithelium (n=76; p<0.0001), suggesting that increased EpCAM expressionrepresents an early event in the development of prostate cancer(Poczatek, J Urol., 1999, 162, 1462-1644). In addition, in the majorityof both squamous and adenocarcinomas of the cervix a strong EpCAMexpression correlates with an increased proliferation and thedisappearance of markers for terminal differentiation (Litvinov, Am. J.Pathol. 1996, 148, 865-75). One example is breast cancer whereoverexpression of EpCAM on tumor cells is a predictor of survival(Gastl, Lancet. 2000, 356, 1981-1982). Furthermore, EpCAM has beendescribed as a marker for the detection of disseminated tumor cells inpatients suffering from squamous cell carcinoma of the head, neck andlung (Chaubal, Anticancer Res 1999, 19, 2237-2242, Piyathilake, HumPathol. 2000, 31, 482-487). Normal squamous epithelium, as found inepidermis, oral cavity, epiglottis, pharynx, larynx and esophagus didnot significantly express. EpCAM (Quak, Hybridoma, 1990, 9, 377-387).

In addition to the above-mentioned carcinomas, EpCAM has been shown tobe expressed on the majority of primary, metastatic, and disseminatedNSCLC (non small cell lung cancer cells) (Passlick, Int J Cancer, 2000,87, 548-552), on gastric and gastro-oesophageal junction adenocarcinomas(Martin, J Clin Pathol 1999, 52, 701-4) and in cell lines derived fromcolorectal, pancreatic carcinomas and breast carcinomas (Szala, ProcNatl Acad Sci USA 1990, 87, 3542-6, Packeisen, Hybridoma, 1999, 18,37-40).

Clinical trials have shown that the use of antibodies directed against17-1A (EpCAM) for treatment of patients with surgically completelyresected colorectal carcinoma leads to a significant benefit concerningthe overall survival and the frequency of distant metastasis(Riethmüller, Lancet, 1994, 343, 1177-1183). Murine monoclonal antibodyagainst EpCAM was found to reduce the 5-year mortality (Riethmüller,Lancet, 1994, 343, 1177-1183) and also the 7-year mortality(Riethmüller, Proceedings of the American Society of Clinical Oncology,1996, 15, 444) of patients with minimal residual disease. Example ofmurine monoclonal antibody recognizing EpCAM is Edrecolomab (Panorex)(Koprowski, Somatic Cell Genet. 1979, 5, 957-971 and Herlyn, CancerRes., 1980, 40, 717-721). However, the first administration of Panorexduring adjuvant immunotherapy of colon cancer led to the development andexacerbation of Wegener's granulomatosis suggesting that mAb 17-1Ashould be applied cautiously in a patient with autoimmune disease(Franz, Onkologie, 2000, 23, 472-474). The limitations of Panorex arethe rapid formation of human anti-mouse antibodies (HAMA), the limitedability to interact by its murine IgG2a Fc-portion with human immuneeffector mechanisms and the short half-life in circulation (Frodin,Cancer Res., 1990, 50, 4866-4871). Furthermore, the murine antibodycaused immediate-type allergic reactions and anaphylaxis upon repeatedinjection in patients (Riethmüller, Lancet. 1994, 343, 1177-1183,Riethmüller, J Clin Oncol., 1998, 16, 1788-1794 and Mellstedt, AnnalsNew York Academy of Sciences. 2000, 910, 254-261).

Humanized anti-EpCAM antibody called 3622W94 resulted in pancreatitisand increased serum levels of amylase, as being indicative for damage ofpancreas epithelium, which were a dose-limiting toxicity of thishigh-affinity anti-EpCAM monoclonal antibody (LoBuglio, Proceedings ofthe American Society of Clinical Oncology (Abstract). 1997, 1562 andKhor, Proceedings of the American Society of Clinical Oncology(Abstract), 1997, 847).

Bispecific antibodies comprising a region directed against EpCAM and aregion directed against CD3 have also been described. The authors ofMöller & Reisfeld 1991 Cancer Immunol. Immunother. 33:210-216 describethe construction of two different bispecific antibodies by fusing ahybridoma producing monoclonal antibody against EpCAM with either of thetwo hybridomas OKT3 and 9.3. Furthermore, Kroesen, Cancer Research,1995, 55:4409-4415 describe a quadroma bispecific monoclonal antibodiesagainst CD3 (BIS-1) and EpCAM.

Other examples of bispecific antibodies against EpCAM comprise thebispecific antibody, BiUII, (anti-CD3 (rat IgG2b) x anti-EpCAM (mouseIgG2a)) a complete Ig molecule which also binds and activatesFc-receptor positive accessory cells (like monocytes/macrophages, NKcells and dendritic cells) through its Fc-region (Zeidler, J. Immunol.,1999, 163:1247-1252) and an anti-EpCAMxanti-CD3 bispecific antibody inthe arrangement V_(L17-1A)-V_(H17-1A)-V_(Hanti-CD3)-V_(Lanti-CD3) (Mack,Proc. Natl. Acad. Sci., 1995, 92:7021-7025).

In addition, other formats of antibody constructs comprising EpCAM havebeen described; e.g. a bispecific diabody having the structureV_(H anti-CD3)-V_(L anti-EpCAM)-V_(H-anti-EpCAM)-V_(Lanti-CD3)(Helfrich, Int. J. Cancer, 1998, 76:232-239) and a trispecific antibodyhaving two different tumour antigen specificities (two antigen bindingregions which bind two different antigens on a tumour cell) and whichmay have a further specificity for an antigen localized on an effectorcell (DE 195 31 348).

There exist various descriptions in the prior art of using phage displaytechnology to identify antibodies or fragments thereof, whichspecifically bind to the human EpCAM antigen (De Kruif JMB, 1995,248:97-105, WO 99/25818). However, it has been extremely difficult toidentify antibodies against EpCAM, which show cytotoxic activitysufficient for therapeutic applications in a bispecific format.

It is therefore an aim of the present invention to provide a bispecificsingle chain molecule with a binding domain specific for EpCAM withstrong cytotoxic activity mediated by target specific activation of Tcells.

Thus, the technical problem underlying the present invention was toprovide means and methods for the generation of well tolerated andconvenient medicaments for the treatment and or amelioration of tumorousdiseases.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to a composition, preferablya pharmaceutical composition, comprising a bispecific single chainantibody construct, whereby said construct comprises or consists of atleast two binding domains, whereby one of said domains binds to humanEpCAM antigen and a second domain binds to human CD3 antigen, whereinsaid binding domain specific for EpCAM comprises at least one CDR-H3region comprising the amino acid sequence NXD preferably in position 102to 104 of SEQ ID NOs: 80, 88 and 96, or preferably in position 106 to108 of SEQ ID NOs: 84 and 92, wherein X is an aromatic amino acid.

Preferably or alternatively, the present invention relates to acomposition, preferably a pharmaceutical composition, comprising abispecific single chain antibody construct, whereby said constructcomprises or consists of at least two domains, whereby one of said atleast two domains specifically binds to human EpCAM antigen and a seconddomain binds to human CD3 antigen, wherein said binding domain specificfor EpCAM comprises at least one CDR-H3 region of least 9 amino acidresidues and wherein said binding domain specific for EpCAM has a K_(D)value of more than 5×10⁻⁹ M.

In accordance with this invention, the term “pharmaceutical composition”relates to a composition for administration to a patient, preferably ahuman patient. In a preferred embodiment, the pharmaceutical compositioncomprises a composition for parenteral, transdermal, intraluminal,intra-arterial, intrathecal or intravenous administration or for directinjection into the tumor. It is in particular envisaged that saidpharmaceutical composition is administered to a patient via infusion orinjection. Administration of the suitable compositions may be effectedby different ways, e.g., by intravenous, subcutaneous, intraperitoneal,intramuscular, topical or intradermal administration. The pharmaceuticalcomposition of the present invention may further comprise apharmaceutically acceptable carrier. Examples of suitable pharmaceuticalcarriers are well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions, etc. Compositions comprising suchcarriers can be formulated by well known conventional methods. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. A preferred dosage foradministration might be in the range of 0.24 μg to 48 mg, preferably0.24 μg to 24 mg, more preferably 0.24 μg to 2.4 mg, even morepreferably 0.24 μg to 1.2 mg and most preferably 0.24 μg to 240 μg unitsper kilogram of body weight per day. Particularly preferred dosages arerecited herein below. Progress can be monitored by periodic assessment.Dosages will vary but a preferred dosage for intravenous administrationof DNA is from approximately 10⁶ to 10¹² copies of the DNA molecule. Thecompositions of the invention may be administered locally orsystematically. Administration will generally be parenteral, e.g.,intravenous; DNA may also be administered directly to the target site,e.g., by biolistic delivery to an internal or external target site or bycatheter to a site in an artery. In an preferred embodiment, thepharmaceutical composition is administered subcutaneously and in an evenmore preferred embodiment intravenously. Preparations for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishes, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like. In addition, thepharmaceutical composition of the present invention might compriseproteinaceous carriers, like, e.g., serum albumine or immunoglobuline,preferably of human origin. It is envisaged that the pharmaceuticalcomposition of the invention might comprise, in addition to theproteinaceous bispecific single chain antibody constructs or nucleicacid molecules or vectors encoding the same (as described in thisinvention), further biologically active agents, depending on theintended use of the pharmaceutical composition. Such agents might bedrugs acting on the gastro-intestinal system, drugs acting ascytostatica, drugs preventing hyperurikemia, agents such as T-cellco-stimulatory molecules or cytokines, drugs inhibiting immune reactions(e.g. corticosteroids) and/or drugs acting on the circulatory system,e.g. on the blood pressure, known in the art.

Possible indications for administration of the composition(s) of theinvention are tumorous diseases especially epithelial cancers/carcinomassuch as breast cancer, colon cancer, prostate cancer, head and neckcancer, skin cancer, cancers of the genito-urinary tract, e.g. ovarialcancer, endometrial cancer, cervix cancer and kidney cancer, lungcancer, gastric cancer, cancer of the small intestine, liver cancer,pancreas cancer, gall bladder cancer, cancers of the bile duct,esophagus cancer, cancer of the salivatory glands and cancer of thethyroid gland. The administration of the composition(s) of the inventionis especially indicated for minimal residual disease preferably earlysolid tumor, advanced solid tumor or metastatic solid tumor, which ischaracterized by the local and non-local reoccurrence of the tumorcaused by the survival of single cells.

The invention further envisages the co-administration protocols withother compounds, e.g. bispecific antibody constructs, targeted toxins orother compounds, which act via T cells. The clinical regimen forco-administration of the inventive compound(s) may encompassco-administration at the same time, before or after the administrationof the other component.

A possible approach to demonstrate the efficacy/activity of theinventive constructs is an in vivo model like mouse. Suitable models maybe transgenic and chimeric mouse models. Mouse models expressing humanCD3 and human EpCAM, a chimeric mouse model expressing murine CD3 andinto which tumour cells expressing human EpCAM can be transfected andchimeric mouse models comprising nude mice into which human tumoursexpressing EpCAM can be transplanted or tumour cells expressing humanEpCAM can be injected and, additionally, human PBMCs are injected. Theterm “bispecific single chain antibody construct” relates to a constructcomprising two antibody derived binding domains. One of said bindingdomains consists of variable regions (or parts thereof) of an antibody,antibody fragment or derivative thereof, capable of specifically bindingto/interacting with human EpCAM antigen (target molecule 1). The secondbinding domain consists of variable regions (or parts thereof) of anantibody, antibody fragment or derivative thereof, capable ofspecifically binding to/interacting with human CD3 antigen (targetmolecule 2). As will be detailed below, a part of a variable region maybe at least one CDR (“Complementary determining region”), mostpreferably at least the CDR3 region. Said two domains/regions in thesingle chain antibody construct are preferably covalently connected toone another as a single chain. This connection can be effected eitherdirectly (domain 1 [specific for the CD3 antigen]-domain 2 [specific forthe EpCAM antigen] or domain 1 [specific for the EpCAM antigen]-domain 2[specific for the CD3 antigen]) or through an additional polypeptidelinker sequence (domain1-linker sequence-domain2). In the event that alinker is used, this linker is preferably of a length and sequencesufficient to ensure that each of the first and second domains can,independently from one another, retain their differential bindingspecificities. Most preferably and as documented in the appendedexamples, the “bispecific single chain antibody construct” to beemployed in the pharmaceutical composition of the invention is abispecific single chain Fv (scFv). Bispecific single chain molecules areknown in the art and are described in WO 99/54440, Mack, J. Immunol.(1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, CancerImmunol. Immunother., (1997), 45, 193-197, Löffler, Blood, (2000), 95,6, 2098-2103 and Brühl, J. Immunol., (2001), 166, 2420-2426. Aparticularly preferred molecular format of the invention provides apolypeptide construct wherein the antibody-derived region comprises oneV_(H) and one V_(L) region. The intramolecular orientation of theV_(H)-domain and the V_(L)-domain, which are linked to each other by alinker-domain, in the scFv format is not decisive for the recitedbispecific single chain constructs. Thus, scFvs with both possiblearrangements (V_(H)-domain-linker domain-V_(L)-domain;V_(L)-domain-linker domain-V_(H)-domain) are particular embodiments ofthe recited bispecific single chain construct.

The antibody construct may also comprise additional domains, e.g. forthe isolation and/or preparation of recombinantly produced constructs.

A corresponding format for a bispecific single chain antibody constructis described in the appended example 1.

The term “single-chain” as used in accordance with the present inventionmeans that said first and second domain of the bispecific single chainconstruct are covalently linked, preferably in the form of a co-linearamino acid sequence encodable by a single nucleic acid molecule.

The term “binding to/interacting with” as used in the context with thepresent invention defines a binding/interaction of at least two“antigen-interaction-sites” with each other. The term“antigen-interaction-site” defines, in accordance with the presentinvention, a motif of a polypeptide which shows the capacity of specificinteraction with a specific antigen or a specific group of antigens.Said binding/interaction is also understood to define a “specificrecognition”. The term “specifically recognizing” means in accordancewith this invention that the antibody molecule is capable ofspecifically interacting with and/or binding to at least two amino acidsof each of the human target molecule as defined herein. Said termrelates to the specificity of the antibody molecule, i.e. to its abilityto discriminate between the specific regions of the human targetmolecule as defined herein. The specific interaction of theantigen-interaction-site with its specific antigen may result in aninitiation of a signal, e.g. due to the induction of a change of theconformation of the antigen, an oligomerization of the antigen, etc.Further, said binding may be exemplified by the specificity of a“key-lock-principle”. Thus, specific motifs in the amino acid sequenceof the antigen-interaction-site and the antigen bind to each other as aresult of their primary, secondary or tertiary structure as well as theresult of secondary modifications of said structure. The specificinteraction of the antigen-interaction-site with its specific antigenmay result as well in a simple binding of said site to the antigen.

The term “specific interaction” as used in accordance with the presentinvention means that the bispecific single chain construct does not oressentially does not cross-react with (poly)peptides of similarstructures. Cross-reactivity of a panel of bispecific single chainconstruct under investigation may be tested, for example, by assessingbinding of said panel of bispecific single chain construct underconventional conditions (see, e.g., Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,1999) to the (poly)peptide of interest as well as to a number of more orless (structurally and/or functionally) closely related (poly)peptides.Only those antibodies that bind to the (poly)peptide/protein of interestbut do not or do not essentially bind to any of the other (poly)peptidesare considered specific for the (poly)peptide/protein of interest.Examples for the specific interaction of an antigen-interaction-sitewith a specific antigen comprise the specificity of a ligand for itsreceptor. Said definition particularly comprises the interaction ofligands which induce a signal upon binding to its specific receptor.Examples for corresponding ligands comprise cytokines whichinteract/bind with/to its specific cytokine-receptors. Also particularlycomprised by said definition is the binding of anantigen-interaction-site to antigens like antigens of the selectinfamily, integrins and of the family of growth factors like EGF. An otherexample for said interaction, which is also particularly comprised bysaid definition, is the interaction of an antigenic determinant(epitope) with the antigenic binding site of an antibody.

The term “binding to/interacting with” may also relate to aconformational epitope, a structural epitope or a discontinuous epitopeconsisting of two regions of the human target molecules or partsthereof. In context of this invention, a conformational epitope isdefined by two or more discrete amino acid sequences separated in theprimary sequence which come together on the surface of the molecule whenthe polypeptide folds to the native protein (Sela, (1969) Science 166,1365 and Layer, (1990) Cell 61, 553-6).

The term “discontinuous epitope” means in context of the inventionnon-linear epitopes that are assembled from residues from distantportions of the polypeptide chain. These residues come together on thesurface of the molecule when the polypeptide chain folds into athree-dimensional structure to constitute a conformational/structuralepitope.

The constructs of the present invention are also envisaged tospecifically bind to/interact with a conformational/structuralepitope(s) composed of and/or comprising the two regions of the humanCD3 complex described herein or parts thereof as disclosed herein below.

Accordingly, specificity can be determined experimentally by methodsknown in the art and methods as disclosed and described herein. Suchmethods comprise, but are not limited to Western blots, ELISA-, RIA-,ECL-, IRMA-, EIA-tests and peptide scans.

The term “antibody fragment or derivative thereof” relates to singlechain antibodies, or fragments thereof, synthetic antibodies, antibodyfragments, such as Fab, a F(ab₂)′, Fv or scFv fragments etc., or achemically modified derivative of any of these. Antibodies to beemployed in accordance with the invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) (e.g.posttranslational and chemical modifications, such as glycosylation andphosphorylation) known in the art either alone or in combination.Methods for introducing such modifications in the DNA sequenceunderlying the amino acid sequence of an immunoglobulin chain are wellknown to the person skilled in the art; see, e.g., Sambrook (1989), loc.cit.

The term “(poly)peptide” as used herein describes a group of moleculeswhich comprise the group of peptides, as well as the group ofpolypeptides. The group of peptides is consisting of molecules with upto 30 amino acids, the group of polypeptides is consisting of moleculeswith more than 30 amino acids.

The term “antibody fragment or derivative thereof” particularly relatesto (poly)peptide constructs comprising at least one CDR.

Fragments or derivatives of the recited antibody molecules define(poly)peptides which are parts of the above antibody molecules and/orwhich are modified by chemical/biochemical or molecular biologicalmethods. Corresponding methods are known in the art and described interalia in laboratory manuals (see Sambrook et al.; Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition 1989and 3rd edition 2001; Gerhardt et al.; Methods for General and MolecularBacteriology; ASM Press, 1994; Lefkovits; Immunology Methods Manual: TheComprehensive Sourcebook of Techniques; Academic Press, 1997; Golemis;Protein-Protein Interactions: A Molecular Cloning Manual; Cold SpringHarbor Laboratory Press, 2002).

Bispecific antibodies that specifically recognize the EpCAM antigen andthe CD3 antigen are described in the prior art, e.g., in Mack (Proc.Natl. Acad. Sci., 1995, 92:7021-7025).

As mentioned above, the said variable domains comprised in the hereindescribed bispecific single chain constructs are connected by additionallinker sequences. The term “peptide linker” defines in accordance withthe present invention an amino acid sequence by which the amino acidsequences of the first domain and the second domain of the definedconstruct are linked with each other. An essential technical feature ofsuch peptide linker is that said peptide linker does not comprise anypolymerization activity. A particularly preferred peptide linker ischaracterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e.(Gly)4Ser, or polymers thereof, i.e. ((Gly)4Ser)x. The characteristicsof said peptide linker, which comprise the absence of the promotion ofsecondary structures are known in the art and described e.g. inDall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (MolImmunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1),73-80). Also particularly preferred are peptide linkers which compriseless amino acid residues. An envisaged peptide linker with less than 5amino acids can comprise 4, 3, 2 or one amino acids. A particularlypreferred “single” amino acid in context of said “peptide linker” isGly. Accordingly, said peptide linker may consist of the single aminoacid Gly. Furthermore, peptide linkers which also do not promote anysecondary structures are preferred. The linkage of said domains to eachother can be provided by, e.g. genetic engineering, as described in theexamples. Methods for preparing fused and operatively linked bispecificsingle chain constructs and expressing them in mammalian cells orbacteria are well-known in the art (e.g. WO 99/54440, Ausubel, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y. 1989 and 1994 or Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001).

The bispecific single chain antibody constructs described herein aboveand below may be humanized or deimmunized antibody constructs. Methodsfor the humanization and/or deimmunization of (poly))peptides and, inparticular, antibody constructs are known to the person skilled in theart.

Here it was surprisingly found that domains with specificity for theEpCAM antigen, comprising at least one CDR-H3 region comprising theamino acid sequence NXD (asparagine-X-aspartic acid) preferably inposition 102 to 104 of SEQ ID NOs: 80, 88 and 96, or in position 106 to108 of SEQ ID NOs: 84 and 92, wherein X is an aromatic amino acid areparticularly useful in the specific format of a bispecific single chainantibody construct. These bispecific single chain antibody constructsare particularly useful as pharmaceutical compositions since theseconstructs are advantageous over constructs which do not comprise saidamino acids.

Furthermore, it was surprisingly found that domains with specificity forthe EpCAM antigen, comprising at least one CDR-H3 region of at least 9amino acid residues and having a K_(D) value of more than 5×10⁻⁹ M areparticularly useful in the specific format of a bispecific single chainantibody construct. These bispecific single chain antibody construct areparticularly useful as pharmaceutical compositions since theseconstructs are advantageous over constructs of less than 9 amino acidresidues and wherein said binding domain specific for EpCAM has a K_(D)value of less than 5×10⁻⁹ M.

The prior art constructs are characterized by less advantageous EC₅₀values and/or less efficient or complete purifications as shown in theappended examples. It was in particular surprising that the domain ofthe single chain constructs with specificity for the CD3 antigen to beemployed in accordance with the invention are highly bioactive in N- aswell as C-terminal position, wherein in particular arrangements inV_(H(anti-CD3))-V_(L(anti-CD3)) are preferred. The constructs to beemployed in the pharmaceutical composition of the invention arecharacterized by advantageous production and purification properties aswell as by their high bioactivity, i.e. their desired cytotoxicactivity. In particular, when the cytotoxic activity of the constructsof the invention were compared with cytotoxic activity of conventionalM79xanti-CD3 and HD70xanti-CD3 constructs, the constructs of theinvention showed clearly higher bioactivity (FIG. 11B). Thecorresponding high bioactivity is reflected by low to very low EC₅₀values as determined in cytotoxicity tests. The lower the EC₅₀ value ofthe molecule is, the higher cytotoxicity, i.e. the effectivity in thecell lysis, of the construct is higher. On the other hand, the higherthe EC₅₀ value, the less effective the molecule is in inducing celllysis. The term “EC₅₀” corresponds, in context of this invention, toEC₅₀ values as determined according to the methods known in the art andas illustrated in the appended examples: A standard dose-response curveis defined by four parameters: the baseline response (Bottom), themaximum response (Top), the slope, and the drug concentration thatprovokes a response halfway between baseline and maximum (EC₅₀). EC₅₀ isdefined as the concentration of a drug or molecule that provokes aresponse half way between the baseline (Bottom) and maximum response(Top). A lower K_(D) value of the constructs of the invention depictshigher binding affinity. E.g. a low K_(D) of 10⁻⁹ M shows high bindingaffinity of the binding construct. On the other hand a high K_(D) valueof e.g. 10⁻⁶ M relates to lower binding affinity of the binding domainof the construct.

The percentage of cell lysis (i.e. cytotoxic activity) may be determinedby, inter glia, release assays disclosed herein above, for example, ⁵¹Crrelease assays, LDH-release assays and the like. Most preferably, incontext of this invention fluorochrome release assays is employed asillustrated in the appended examples. Here, strong cytotoxic activityagainst EpCAM-positive cells (see CHO-EpCAM cells in appended example 3)of the bispecific single chain constructs described herein relates to amolecule comprising EC₅₀ values preferably ≦500 pg/ml, more preferably≦400 pg/ml, even more preferably ≦300 pg/ml, even more preferably ≦250pg/ml, most preferably ≦200 pg/ml, ≦100 pg/ml, 50 pg/ml.

The bispecific constructs comprised in the pharmaceutical compositionsof the present invention show a surprisingly high cytotoxic activity(preferably in the range of about 10 pg/ml to 170 pg/ml) compared to theprior art M79xanti-CD3 construct(VL_(17-1A)-VH_(17-1A)-VH_(CD3)-VL_(CD3); 8628 pg/ml). A skilled personis aware that EC50 values may vary depending to the bioactivity assay.Factors affecting EC50 value may comprise type of effector cells,activity of effector cells, type of target cells, E:T ratio, incubationtime, incubation temperature and other external circumstances. DifferentEC50 values of same constructs in different experiments may be comparedwith the EC50 values of controls. A construct having high cytotoxicactivity according to the invention has at least 2.5 time lower EC50value than the control (at least 2.5 times higher cytotoxicity than thecontrol), preferably at least three times lower EC50 value and morepreferably at least five times lower EC50 value.

Furthermore, the constructs of the invention bind EpCAM with asurprisingly high affinity measured by surface plasmonresonance)(BIAcore°. The prior art EpCAM and CD3 binding constructM79xanti-CD3 has a K_(D) of 4×10⁻⁶ M and the constructs of the inventiona K_(D) of 2.3×10⁻⁷-2.5×10⁻⁷ M.

Preferably, the X in said NXD motif is W (tryptophan) or Y (tyrosine).

It is further envisaged that the pharmaceutical composition of theinvention comprises a bispecific single chain antibody construct,wherein the CDR-H3 of the EpCAM specific domain comprises at least 9amino acid residues, preferably at least 14 amino acids. Preferably theCDR-H3 comprises less than 18 amino acids, more preferably less than 15amino acids. Thus, preferably the CDR-H3 comprises 9 to 17 amino acids,more preferably 9 to 15 amino acids and most preferably 10 or 14 aminoacids.

Bispecific single chain antibody construct comprising a correspondingEpCAM specific domain have been surprisingly found to be advantagous inthe format of the above described construct over other EpCAM specificdomain known in the art. Such effect is demonstrated in appendedexamples 3, 4 and 5. The prior art EpCAM binding antibody M79 compriseseight amino acids in its CDR-H3 region and does not comprise thesequence NXD (FIG. 11A).

The pharmaceutical composition according to the invention may alsocomprise constructs, wherein said binding domain specific for EpCAM hasa K_(D) value of more than 5×10⁻⁹ M.

The pharmaceutical composition may additionally be characterized by thefeature that said binding domain specific for the CD3 antigen has aK_(D) value of more than 10⁻⁷ M.

The K_(D) value is a physical value defining the tendency of a complexto dissociate. For the binding equilibrium A+B

AB, the dissociation constant is given as the ratio of the two kineticrate constants k_(off) and k_(on): [A][B] (kon)/[AB] (koff). The smallerthe dissociation constant the tighter A and B bind to each other. Inbiological systems a good, specific binder has a dissociation constantin the range of 10⁻⁹-10⁻⁷ M. K_(D) can be measured with a number ofmethods known to the person skilled in the art, e.g. surface plasmonresonance (SPR, e.g. with BIAcore®), analytical ultracentrifugation,isothermal titration calorimetry, fluorescence anisotropy, fluorescencespectroscopy or by radiolabeled ligand binding assays.

The K_(D)s of the constructs of the invention have been measured usingthe surface plasmon resonance (SPR) spectroscopy. The ligand is injectedover the immobilized antigen chip surface and the change in opticaldensity on the chip surface upon binding is measured. The change inoptical density, monitored by a change in reflection angle, correlatesdirectly to the amount of ligand binding to the chip surface—thebiophysical phenomenon used is called surface plasmon resonance.

One of the interaction partners has to be immobilized on the surface ofthe sensor chip of the apparatus based on surface plasmon resonance(e.g. BIAcore®). The kinetics of association and dissociation of ligandwith the immobilized antigen on the chip surface are observed in realtime. The binding curves are fitted for kinetic rate constants k_(on)and k_(off), resulting in an apparent equilibrium dissociation constant(KD).

It is particularly preferred, that said binding domain specific forEpCAM has a K_(D) value in a range between 1×10⁻⁷ and 5×10⁻⁹ M and saidbinding domain specific for CD3 has a K_(D) value in a range between1×10⁻⁶ and 5×10⁻⁹ M.

In a particularly preferred embodiment, the pharmaceutical compositionmay additionally be characterized by the feature that said bindingdomain specific for the CD3 antigen has a K_(D) value of >(more than)1×10⁻⁷ M.

The constructs of the invention have the advantage that they may be useda number of times for killing tumour cells since the EpCAM binding parthas an affinity with a K_(D) value of more than 5×10⁻⁹ M. If theaffinity of a bispecific construct for binding an EpCAM-expressingtumour cell is too high, the construct binds one EpCAM expressing tumourcell and remains on its surface even when it has been killed and cannotcontinue to another tumour cell to be killed. A further advantage of theconstruct of the invention is that the binding domain specific for EpCAMbinds with a high affinity (corresponds to lower K_(D) value), thusleading the circulating T-cells to the tumour cells marked with thebispecific construct. Therefore, the K_(D) of the binding domainspecific for EpCAM of the bispecific construct is preferably in therange of 10⁻⁷-5×10⁻⁹ M and the K_(D) of the binding domain specific forCD3 is preferably in the range of 10⁻⁶-5×10⁻⁹ M. In a preferredembodiment, the KD value of the EpCAM binding domain is lower than theKD value of the CD3 binding domain corresponding to a higher affinity ofthe EpCAM binding domain compared to the CD3 binding domain.

Further it is envisaged that the pharmaceutical composition of theinvention comprises a bispecific single chain antibody construct,wherein the CDR-H3 of the EpCAM specific domain comprises at least 9amino acids, preferably at least 14 amino acids. Preferably the CDR-H3comprises less than 18 amino acids, more preferably less than 15 aminoacids. Thus, preferably the CDR-H3 comprises 9 to 17 amino acids, morepreferably 9 to 15 amino acids and most preferably 10 or 14 amino acids.

In a preferred embodiment of the pharmaceutical composition of theinvention the V_(H) chain of the domain specific for human EpCAM antigenis selected from the group consisting of:

-   (a) an amino acid sequence as shown in any of SEQ ID NO: 80, SEQ ID    NO: 84, SEQ ID NO: 88, SEQ ID NO: 92 and SEQ ID NO:96;-   (b) an amino acid sequence encoded by a nucleic acid sequence as    shown in SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91    and SEQ ID NO: 95;-   (c) an amino acid sequence encoded by a nucleic acid sequence    hybridizing with the complementary strand of a nucleic acid sequence    as defined in (b) under stringent hybridization conditions;-   (d) an amino acid sequence encoded by a nucleic acid sequence which    is degenerate as a result of the genetic code to a nucleotide    sequence of any one of (b) and (c).

The term “hybridizing” as used herein refers to polynucleotides/nucleicacid sequences which are capable of hybridizing to the polynucleotidesencoding bispecific single chain constructs as defined herein or partsthereof. Therefore, said polynucleotides may be useful as probes inNorthern or Southern Blot analysis of RNA or DNA preparations,respectively, or can be used as oligonucleotide primers in PCR analysisdependent on their respective size. Preferably, said hybridizingpolynucleotides comprise at least 10, more preferably at least 15nucleotides in length while a hybridizing polynucleotide of the presentinvention to be used as a probe preferably comprises at least 100, morepreferably at least 200, or most preferably at least 500 nucleotides inlength.

It is well known in the art how to perform hybridization experimentswith nucleic acid molecules, i.e. the person skilled in the art knowswhat hybridization conditions s/he has to use in accordance with thepresent invention. Such hybridization conditions are referred to instandard text books such as Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory (2001) N.Y. Preferred in accordance with thepresent inventions are polynucleotides which are capable of hybridizingto the polynucleotides of the invention or parts thereof, understringent hybridization conditions.

“Stringent hybridization conditions” refer, e.g. to an overnightincubation at 42° C. in a solution comprising 50% formamide, 5×SSC (750mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C. Also contemplated are nucleic acid molecules that hybridizeto the polynucleotides of the invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37° C. in asolution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA;followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, toachieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC). Itis of note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

The recited nucleic acid molecules may be, e.g., DNA, cDNA, RNA orsynthetically produced DNA or RNA or a recombinantly produced chimericnucleic acid molecule comprising any of those polynucleotides eitheralone or in combination.

Preferably said pharmaceutical composition of the invention may comprisea bispecific single chain construct, wherein the V_(L) chain domainsspecific for human EpCAM antigen is selected from the group consistingof:

-   (a) an amino acid sequence as shown in any of SEQ ID NO: 82, SEQ ID    NO: 86, SEQ ID NO: 90, SEQ ID NO: 94 and SEQ ID NO: 98;-   (b) an amino acid sequence encoded by a nucleic acid sequence as    shown in SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93    and SEQ ID NO: 97;-   (c) an amino acid sequence encoded by a nucleic acid sequence    hybridizing with the complementary strand of a nucleic acid sequence    as defined in (b) under stringent hybridization conditions;-   (d) an amino acid sequence encoded by a nucleic acid sequence which    is degenerate as a result of the genetic code to a nucleotide    sequence of any one of (b) and (c).

In a preferred embodiment of the pharmaceutical composition of thisinvention, the V_(H) and V_(L) regions of said human CD3 specific domainare derived from an CD3 specific antibody selected from the groupconsisting of X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5,F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46,XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, WT31 andF101.01. These CD3-specific antibodies are well known in the art and,inter alia, described in Tunnacliffe (1989), Int. Immunol. 1, 546-550.In a more preferred embodiment, said V_(H) and V_(L) regions of said CD3specific domain are derived from OKT-3 (as defined and described above).Even more preferred. (and as illustrated in the appended examples) saidV_(H) and V_(L) regions are or are derived from an antibody/antibodyderivative with specificity for the CD3 molecule described by Traunecker(1991), EMBO J. 10, 3655-3659. In accordance with this invention, saidV_(H) and V_(L) regions are derived from antibodies/antibody derivativesand the like which are capable of specifically recognizing the humanCD3-ε chain in the context of other TCR subunits, e.g. in mouse cellstransgenic for human CD3-ε chain. These transgenic mouse cells expresshuman CD3-ε chain in a native or near native conformation. Accordingly,the V_(H) and V_(L) regions derived from an CD3-ε chain specificantibody is most preferred in accordance with this invention and said(parental) antibodies should be capable of specifically binding epitopesreflecting the native or near native structure or a conformationalepitope of human CD3 presented in context of the TCR complex. Suchantibodies have been classified by Tunnacliffe (1989) as “group II”antibodies. Further classifications in Tunnacliffe (1989) comprise thedefinition of “group I” and “group III” antibodies directed against CD3.“Group I” antibodies, like UCHT1, recognize CD3-ε chain expressed asrecombinant protein and as part of the TCR on the cell surface.Therefore, “group I” antibodies are highly specific for CD3-ε chain. Incontrast, the herein preferred “group II antibodies” recognize CD3-εchain only in the native TCR complex in association with other TCRsubunits. Without being bound by theory, it is speculated in context ofthis invention that in “group II” antibodies, the TCR context isrequired for recognition of CD3-ε chain. CD3-γ chain and δ chain, beingassociated with ε chain, are also involved in binding of “group IIantibodies”. All three subunits express immunoreceptor-tyrosineactivation motifs (ITAMs) which can be tyrosine phosphorylated byprotein tyrosine-based kinases. For this reason group II antibodiesinduce T cell signaling via CD3-ε chain, γ chain and δ chain, leading toa stronger signal compared to group I antibodies selectively inducing Tcell signaling via CD3-ε chain. Yet, since for therapeutic applicationsinduction of a strong T cell signaling is desired, theV_(H (anti-CD3))/V_(L (anti-CD3))-regions (or parts thereof) to beemployed in the bispecific single chain constructs comprised in theinventive pharmaceutical composition, are preferably derived fromantibodies directed against human CD3 and classified in “group II” byTunnacliffe (1989), loc.cit.

In one embodiment the present invention relates to a pharmaceuticalcomposition wherein said bispecific single chain antibody constructcomprises an amino acid sequence selected from the group of:

-   (a) an amino acid sequence as shown in any of SEQ ID NOs: 2, 4, 8,    10, 12, 14, 16, 18, 20, 30, 36, 39, 42, 44, 46, 48, 50, 52, 54, 56,    58 and 60;-   (b) an amino acid sequence encoded by a nucleic acid sequence as    shown in any of SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15, 17, 19, 29, 35,    38, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59;-   (c) an amino acid sequence encoded by a nucleic acid sequence    hybridizing with the complementary strand of a nucleic acid sequence    as defined in (b) under stringent hybridization conditions;-   (d) an amino acid sequence encoded by a nucleic acid sequence which    is degenerate as a result of the genetic code to a nucleotide    sequence of any one of (b) and (c).

The present invention also provides for a pharmaceutical compositioncomprising a nucleic acid sequence encoding a bispecific single chainantibody construct as defined above.

Said nucleic acid molecule may be a natural nucleic acid molecule aswell as a recombinant nucleic acid molecule. The nucleic acid moleculemay, therefore, be of natural origin, synthetic or semi-synthetic. Itmay comprise DNA, RNA as well as PNA (peptide nucleic acid) and it maybe a hybrid thereof.

Thus, the present invention relates to a pharmaceutical compositioncomprising a nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of:

-   (a) a nucleotide sequence encoding the mature form of a protein    comprising the amino acid sequence of the bispecific single chain    antibody constructs defined herein, preferably as given in SEQ ID    Nos: 2, 4, 8, 10, 12, 14, 16, 18, 20, 30, 36, 39, 42, 44, 46, 48,    50, 52, 54, 56, 58 and 60;-   (b) a nucleotide sequence comprising or consisting of the DNA    sequence as given in SEQ ID Nos: 1, 3, 7, 9, 11, 13, 15, 17, 19, 29,    35, 38, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59;-   (c) a nucleotide sequence hybridizing with the complementary strand    of a nucleotide sequence as defined in (b) under stringent    hybridization conditions;-   (d) a nucleotide sequence encoding a protein derived from the    protein encoded by a nucleotide sequence of (a) or (b) by way of    substitution, deletion and/or addition of one or several amino acids    of the amino acid sequence encoded by the nucleotide sequence of (a)    or (b);-   (e) a nucleotide sequence encoding a protein having an amino acid    sequence at least 60% identical to the amino acid sequence encoded    by the nucleotide sequence of (a) or (b);-   (f) a nucleotide sequence which is degenerate as a result of the    genetic code to a nucleotide sequence of any one of (a) to (e);

The term “mature form of the protein” defines in context with thepresent invention a protein translated from its corresponding mRNA andoptional subsequently modified.

The term “hybridizing” has been defined in the context of the presentinvention herein above.

It is evident to the person skilled in the art that regulatory sequencesmay be added to the nucleic acid molecule comprised in thepharmaceutical composition of the invention. For example, promoters,transcriptional enhancers and/or sequences which allow for inducedexpression of the polynucleotide of the invention may be employed. Asuitable inducible system is for example tetracycline-regulated geneexpression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad.Sci. USA 89 (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12(1994), 58-62), or a dexamethasone-inducible gene expression system asdescribed, e.g. by Crook (1989) EMBO J. 8, 513-519.

Furthermore, it is envisaged for further purposes that nucleic acidmolecules may contain, for example, thioester bonds and/or nucleotideanalogues. Said modifications may be useful for the stabilization of thenucleic acid molecule against endo- and/or exonucleases in the cell.Said nucleic acid molecules may be transcribed by an appropriate vectorcontaining a chimeric gene which allows for the transcription of saidnucleic acid molecule in the cell. In this respect, it is also to beunderstood that such polynucleotide can be used for “gene targeting” or“gene therapeutic” approaches. In another embodiment said nucleic acidmolecules are labeled. Methods for the detection of nucleic acids arewell known in the art, e.g., Southern and Northern blotting, PCR orprimer extension. This embodiment may be useful for screening methodsfor verifying successful introduction of the nucleic acid moleculesdescribed above during gene therapy approaches.

Said nucleic acid molecule(s) may be a recombinantly produced chimericnucleic acid molecule comprising any of the aforementioned nucleic acidmolecules either alone or in combination. Preferably, the nucleic acidmolecule is part of a vector.

The present invention therefore also relates to a pharmaceuticalcomposition comprising a vector comprising the nucleic acid moleculedescribed in the present invention.

Many suitable vectors are known to those skilled in molecular biology,the choice of which would depend on the function desired and includeplasmids, cosmids, viruses, bacteriophages and other vectors usedconventionally in genetic engineering. Methods which are well known tothose skilled in the art can be used to construct various plasmids andvectors; see, for example, the techniques described in Sambrook et al.(loc cit.) and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), (1994).Alternatively, the polynucleotides and vectors of the invention can bereconstituted into liposomes for delivery to target cells. As discussedin further details below, a cloning vector was used to isolateindividual sequences of DNA. Relevant sequences can be transferred intoexpression vectors where expression of a particular polypeptide isrequired. Typical cloning vectors include pBluescript SK, pGEM, pUC9,pBR322 and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK,pESP-1, pOP13CAT.

Preferably said vector comprises a nucleic acid sequence which is aregulatory sequence operably linked to said nucleic acid sequenceencoding a bispecific single chain antibody constructs defined herein.

Such regulatory sequences (control elements) are known to the artisanand may include a promoter, a splice cassette, translation initiationcodon, translation and insertion site for introducing an insert into thevector. Preferably, said nucleic acid molecule is operatively linked tosaid expression control sequences allowing expression in eukaryotic orprokaryotic cells.

It is envisaged that said vector is an expression vector comprising thenucleic acid molecule encoding a bispecific single chain antibodyconstructs defined herein.

The term “regulatory sequence” refers to DNA sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, control sequences generally includepromoters, ribosomal binding sites, and terminators. In eukaryotesgenerally control sequences include promoters, terminators and, in someinstances, enhancers, transactivators or transcription factors. The term“control sequence” is intended to include, at a minimum, all componentsthe presence of which are necessary for expression, and may also includeadditional advantageous components.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. In case the control sequence is a promoter, it is obvious fora skilled person that double-stranded nucleic acid is preferably used.

Thus, the recited vector is preferably an expression vector. An“expression vector” is a construct that can be used to transform aselected host and provides for expression of a coding sequence in theselected host. Expression vectors can for instance be cloning vectors,binary vectors or integrating vectors. Expression comprisestranscription of the nucleic acid molecule preferably into atranslatable mRNA. Regulatory elements ensuring expression inprokaryotes and/or eukaryotic cells are well known to those skilled inthe art. In the case of eukaryotic cells they comprise normallypromoters ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Possible regulatory elements permitting expression inprokaryotic host cells comprise, e.g., the P_(L), lac, trp or tacpromoter in E. coli, and examples of regulatory elements permittingexpression in eukaryotic host cells are the AOX1 or GAL1 promoter inyeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus),CMV-enhancer, SV40-enhancer or a globin intron in mammalian and otheranimal cells.

Beside elements which are responsible for the initiation oftranscription such regulatory elements may also comprise transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Furthermore, depending on theexpression system used leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediummay be added to the coding sequence of the recited nucleic acid sequenceand are well known in the art; see also, e.g., the appended examples.The leader sequence(s) is (are) assembled in appropriate phase withtranslation, initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated protein, ora portion thereof, into the periplasmic space or extracellular medium.Optionally, the heterologous sequence can encode a fusion proteinincluding an N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product; see supra. In this context, suitableexpression vectors are known in the art such as Okayama-Berg cDNAexpression vector pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc/CMV, pcDNA1,pcDNA3 (In-vitrogene), pEF-DHFR and pEF-ADA, (Raum et al. Cancer ImmunolImmunother (2001) 50(3), 141-150) or pSPORT1 (GIBCO BRL).

Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming of transfecting eukaryotichost cells, but control sequences for prokaryotic hosts may also beused. Once the vector has been incorporated into the appropriate host,the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and as desired, the collectionand purification of the polypeptide of the invention may follow; see,e.g., the appended examples.

An alternative expression system which could be used to express a cellcycle interacting protein is an insect system. In one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes in Spodoptera frugiperda cells or inTrichoplusia larvae. The coding sequence of a recited nucleic acidmolecule may be cloned into a nonessential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of said coding sequence will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein coat. The recombinant viruses are then used to infect S.frugiperda cells or Trichoplusia larvae in which the protein of theinvention is expressed (Smith, J. Virol. 46 (1983), 584; Engelhard,Proc. Nat. Acad. Sci. USA 91 (1994), 3224-3227).

Additional regulatory elements may include transcriptional as well astranslational enhancers. Advantageously, the above-described vectors ofthe invention comprises a selectable and/or scorable marker.

Selectable marker genes useful for the selection of transformed cellsand, e.g., plant tissue and plants are well known to those skilled inthe art and comprise, for example, antimetabolite resistance as thebasis of selection for dhfr, which confers resistance to methotrexate(Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, whichconfers resistance to the aminoglycosides neomycin, kanamycin andparomycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, whichconfers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485).Additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman, Proc.Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerasewhich allows cells to utilize mannose (WO 94/20627) and ODC (ornithinedecarboxylase) which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory ed.) or deaminase from Aspergillus terreus which confersresistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59(1995), 2336-2338).

Useful scorable markers are also known to those skilled in the art andare commercially available. Advantageously, said marker is a geneencoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J.Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett.389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6 (1987),3901-3907). This embodiment is particularly useful for simple and rapidscreening of cells, tissues and organisms containing a recited vector.

As described above, the recited nucleic acid molecule can be used aloneor as part of a vector to express the encoded polypeptide in cells, for,e.g., gene therapy. The nucleic acid molecules or vectors containing theDNA sequence(s) encoding any one of the above described bispecificsingle chain antibody constructs is introduced into the cells which inturn produce the polypeptide of interest. Gene therapy, which is basedon introducing therapeutic genes into cells by ex-vivo or in-vivotechniques is one of the most important applications of gene transfer.Suitable vectors, methods or gene-delivery systems for in-vitro orin-vivo gene therapy are described in the literature and are known tothe person skilled in the art; see, e.g., Giordano, Nature Medicine 2(1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson,Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239; Isner,Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086;Onodera, Blood 91 (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699;Nabel, Ann. N.Y. Acad. Sci. 811 (1997), 289-292; Verzeletti, Hum. GeneTher. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996), 714-716; WO94/29469; WO 97/00957, U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,589,466;or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. Therecited nucleic acid molecules and vectors may be designed for directintroduction or for introduction via liposomes, or viral vectors (e.g.,adenoviral, retroviral) into the cell. Preferably, said cell is a germline cell, embryonic cell, or egg cell or derived therefrom, mostpreferably said cell is a stem cell. An example for an embryonic stemcell can be, inter alia, a stem cell as described in, Nagy, Proc. Natl.Acad. Sci. USA 90 (1993), 8424-8428.

In accordance with the above, the present invention relates to methodsto derive vectors, particularly plasmids, cosmids, viruses andbacteriophages used conventionally in genetic engineering that comprisea nucleic acid molecule encoding the polypeptide sequence of abispecific single chain antibody constructs defined herein. Preferably,said vector is an expression vector and/or a gene transfer or targetingvector. Expression vectors derived from viruses such as retroviruses,vaccinia virus, adeno-associated virus, herpes viruses, or bovinepapilloma virus, may be used for delivery of the recited polynucleotidesor vector into targeted cell populations. Methods which are well knownto those skilled in the art can be used to construct recombinantvectors; see, for example, the techniques described in Sambrook et al.(loc cit.), Ausubel (1989, loc cit.) or other standard text books.Alternatively, the recited nucleic acid molecules and vectors can bereconstituted into liposomes for delivery to target cells. The vectorscontaining the nucleic acid molecules of the invention can betransferred into the host cell by well-known methods, which varydepending on the type of cellular host. For example, calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment or electroporation may be used for other cellularhosts; see Sambrook, supra.

The recited vector may be the pEF-DHFR, pEF-ADA or pEF-neo.

The vectors pEF-DHFR and pEF-ADA have been described in the art, e.g. inMack et al. (PNAS (1995) 92, 7021-7025) and Ram et al. (Cancer ImmunolImmunother (2001) 50(3), 141-150).

It is further envisaged that the pharmaceutical composition of theinvention comprises a host transformed or transfected with a vectordefined herein above.

Said host may be produced by introducing said at least one of the abovedescribed vector or at least one of the above described nucleic acidmolecules into the host. The presence of said at least one vector or atleast one nucleic acid molecule in the host may mediate the expressionof a gene encoding the above described bespecific single chan antibodyconstructs.

The described nucleic acid molecule or vector which is introduced in thehost may either integrate into the genome of the host or it may bemaintained extrachromosomally.

The host can be any prokaryote or eukaryotic cell.

The term “prokaryote” is meant to include all bacteria which can betransformed or transfected with a DNA or RNA molecules for theexpression of a protein of the invention. Prokaryotic hosts may includegram negative as well as gram positive bacteria such as, for example, E.coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Theterm “eukaryotic” is meant to include yeast, higher plant, insect andpreferably mammalian cells. Depending upon the host employed in arecombinant production procedure, the protein encoded by thepolynucleotide of the present invention may be glycosylated or may benon-glycosylated. Especially preferred is the use of a plasmid or avirus containing the coding sequence of the polypeptide of the inventionand genetically fused thereto an N-terminal FLAG-tag and/or C-terminalHis-tag. Preferably, the length of said FLAG-tag is about 4 to 8 aminoacids, most preferably 8 amino acids. An above described polynucleotidecan be used to transform or transfect the host using any of thetechniques commonly known to those of ordinary skill in the art.Furthermore, methods for preparing fused, operably linked genes andexpressing them in, e.g., mammalian cells and bacteria are well-known inthe art (Sambrook, loc cit.).

Preferably, said the host is a bacteria, an insect, fungal, plant oranimal cell.

It is particularly envisaged that the recited host may be a mammaliancell, more preferably a human cell or human cell line.

Particularly preferred host cells comprise CHO cells, COS cells, myelomacell lines like SP2/0 or NS/0.

The pharmaceutical composition of the invention may also comprise aproteinaceous compound capable of providing an activation signal forimmune effector cells useful for cell proliferation or cell stimulation.

The proteinaceous compound is not understood as an additional domain ofthe above defined bispecific single chain antibody construct, but atleast one additional component of the pharmaceutical composition of theinvention.

In the light of the present invention, said “proteinaceous compounds”providing an activation signal for immune effector cells” may be, e.g. afurther activation signal for T cells (e.g. a further costimulatorymolecule: molecules of the B7-family, Ox40 L, 4.1 BBL), or a furthercytokine: interleukin (e.g. IL-2), or an NKG-2D engaging compound.Preferred formats of proteinaceous compounds comprise additionalbispecific antibodies and fragments or derivatives thereof, e.g.bispecific scFv. Proteinaceous compounds can comprise, but are notlimited to scFv fragments specific for the T cell receptor orsuperantigens. Superantigens directly bind to certain subfamilies of Tcell receptor variable regions in an MHC-independent manner thusmediating the primary T cell activation signal. The proteinaceouscompound may also provide an activation signal for immune effector cellwhich is a non-T cell. Examples for immune effector cells which arenon-T cells comprise, inter alia, NK cells.

An additional technical feature of the pharmaceutical composition of theinvention is that said pharmaceutical composition is thermostable at≧37° C.

An alternative embodiment of the invention relates to a process for theproduction of a pharmaceutical composition of the invention, saidprocess comprising culturing a host defined herein above underconditions allowing the expression of the construct and recovering theproduced bispecific single chain antibody construct from the culture.

The transformed hosts can be grown in fermentors and cultured accordingto techniques known in the art to achieve optimal cell growth. Thepolypeptide of the invention can then be isolated from the growthmedium, cellular lysates, or cellular membrane fractions. The isolationand purification of the, e.g., microbially expressed polypeptides of theinvention may be by any conventional means such as, for example,preparative chromatographic separations and immunological separationssuch as those involving the use of monoclonal or polyclonal antibodiesdirected, e.g., against a tag of the polypeptide of the invention or asdescribed in the appended examples.

The conditions for the culturing of a host which allow the expressionare known in the art and discussed herein above. The same holds true forprocedures for the purification/recovery of said constructs.

A further alternative embodiment of the invention relates to the use ofa bispecific single chain antibody construct as defined above, a nucleicacid sequence as defined above, a vector as defined above, a host asdefined above and/or produced by a process as defined above for thepreparation of a pharmaceutical composition for the prevention,treatment or amelioration of a tumorous disease.

In particular, the pharmaceutical composition of the present inventionmay be particularly useful in preventing, ameliorating and/or treatingcancer.

Preferably said tumorous disease is epithelial cancer or a minimalresidual cancer.

It is envisaged by the present invention that the above definedbispecific single chain antibody construct, nucleic acid molecules andvectors are administered either alone or in any combination usingstandard vectors and/or gene delivery systems, and optionally togetherwith a pharmaceutically acceptable carrier or excipient. Subsequent toadministration, said nucleic acid molecules or vectors may be stablyintegrated into the genome of the subject.

On the other hand, viral vectors may be used which are specific forcertain cells or tissues and persist in said cells. Suitablepharmaceutical carriers and excipients are well known in the art. Thepharmaceutical compositions prepared according to the invention can beused for the prevention or treatment or delaying the above identifieddiseases.

Furthermore, it is possible to use a pharmaceutical composition of theinvention which comprises described nucleic acid molecules or vectors ingene therapy. Suitable gene delivery systems may include liposomes,receptor-mediated delivery systems, naked DNA, and viral vectors such asherpes viruses, retroviruses, adenoviruses, and adeno-associatedviruses, among others. Delivery of nucleic acids to a specific site inthe body for gene therapy may also be accomplished using a biolisticdelivery system, such as that described by Williams (Proc. Natl. Acad.Sci. USA 88 (1991), 2726-2729). Further methods for the delivery ofnucleic acids comprise particle-mediated gene transfer as, e.g.,described in Verma, Gene Ther. 15 (1998), 692-699.

Furthermore the invention relates to a method for the prevention,treatment or amelioration of a tumorous disease comprising the step ofadministering to a subject in the need thereof an effective amount abispecific single chain antibody construct as defined above, a nucleicacid sequence as defined above, a vector as defined as defined above, ahost as defined above and/or produced in by a process as defined above.

Preferably said subject is a human.

The method for the prevention, treatment or amelioration of theinvention may comprise the co-administration of an above definedproteinaceous compound capable of an activation signal for immuneeffector cells to the subject. The co-administration may be asimultaneous co-administration or a non-simultaneous co-administration.

It is particularly preferred for the use and the method of the inventionthat said tumorous disease is epithelial cancer, preferablyadenocarcinomas, or a minimal residual cancer, preferably early solidtumor, advanced solid tumor or metastatic solid tumor.

Finally, the present invention relates to a kit comprising a bispecificsingle chain antibody construct as defined above, a nucleic acidsequence as defined above, a vector as defined above and/or a host asdefined above. It is also envisaged that the kit of this inventioncomprises a pharmaceutical composition as described herein above, eitheralone or in combination with further medicaments to be administered to apatient in need of medical treatment or intervention.

The Figures show:

FIG. 1:

DNA and amino acid sequence of the anti-CD3-anti-EpCAM constructs A)anti-CD3 VHVL stL x 3-1 VHVL (SEQ ID NO.:11,12), B) anti-CD3 VHVL aL x4-7 VHVL (SEQ ID NO.:1,2), C) anti-CD3 VHVL aL Ser x 4-7 VHVL (SEQ IDNO.:7, 8), D) anti-CD3 VHVL stL x 4-7 VHVL (SEQ ID NO.:13,14), E)anti-CD3 VHVL stL x 4-7 VLVH (SEQ ID NO.:15,16), F) anti-CD3 VHVL aL x5-10 VHVL (SEQ ID NO.:3,4), G) anti-CD3 VHVL aL Ser x 5-10 VHVL (SEQ IDNO.:9, 10), H) anti-CD3 VHVL stL x 5-10 VHVL (SEQ ID NO.:17,18), I)anti-CD3 VHVL stL x 5-10 VLVH (SEQ ID NO.:19,20), J) anti-CD3 VHVL aL x3-1 VHVL (SEQ ID NO.:45, 46), K) anti-CD3 VHVL aL Ser x 3-1 VHVL (SEQ IDNO.:47,48), L) anti-CD3 VHVL aL x 3-5 VHVL (SEQ ID NO.:49,50), M)anti-CD3 VHVL aL Ser x 3-5 VHVL (SEQ ID NO.:51,52), N) anti-CD3 VHVL stLx 3-5 VHVL (SEQ ID NO.:53,54), O) anti-CD3 VHVL aL x 4-1 VHVL (SEQ IDNO.:55,56), P) anti-CD3 VHVL aL Ser x 4-1 VHVL (SEQ ID NO.:57,58) and Q)anti-CD3 VHVL stL x 4-1 VHVL (SEQ ID NO.:59,60).

FIG. 2:

FACS analysis of the constructs A) anti-CD3 VHVL stL x 5-10 VHVL (SEQ IDNO.:18), B) anti-CD3 VHVL stL x 4-7 VHVL (SEQ ID NO.:14), C) anti-CD3VHVL aL x 5-10 VHVL (SEQ ID NO.:4), D) anti-CD3 VHVL aL x 4-7 VHVL (SEQID NO.:2), E) anti-CD3 VHVL aL Ser x 5-10 VHVL (SEQ ID NO.:10), F)anti-CD3 VHVL aL Ser x 4-7 VHVL (SEQ ID NO.:8), G) anti-CD3 VHVL stL x3-1 VHVL (SEQ ID NO.:12), H) anti-CD3 VHVL stL x 5-10 VLVH (SEQ IDNO.:20) and I) anti-CD3 VHVL stL x 4-7 VLVH (SEQ ID NO.:16) in CD3positive Jurkat and EpCAM-positive Kato III cells. A shift to the rightshows binding. In Jurkat and KatoIII cells the dotted line indicates theshift of the negative control (only secondary antibody), dashed lineshows the binding of an anti-EpCAM-anti-CD3 control antibody and thebold line shows the bispecific construct of interest.

FIG. 3:

DNA and amino acid sequence of the anti-EpCAM-anti-CD3-constructs A) 4-7VLVHx anti-CD3 VHVL (SEQ ID NO.:41,42), B) 3-5 VLVHx anti-CD3 VHVL (SEQID NO.:29,30), C) 3-1 VLVHx anti-CD3 VHVL (SEQ ID NO.:35,36), D) 4-1VLVHx anti-CD3 VHVL (SEQ ID NO.:38,39) and E) 5-10 VLVHx anti-CD3 VHVL(SEQ ID NO.:43,44).

FIG. 4: FACS analysis of the constructs A) 4-7 VLVHx anti-CD3 VHVL (SEQID NO.:42), B) 3-5 VLVHx anti-CD3 VHVL (SEQ ID NO.:30), C) 3-1 VLVHxanti-CD3 VHVL (SEQ ID NO.:36), D) 4-1 VLVHx anti-CD3 VHVL (SEQ IDNO.:39) and E) 5-10 VLVHx anti-CD3 VHVL (SEQ ID NO.: 44) constructs inCD3 positive Jurkat and EpCAM-positive Kato III cells. A shift to theright shows binding.

FIG. 5:

A representative elution pattern of an EpCAM bispecific antibodycontaining protein fractions from a Zn-Chelating Fractogel® column at280 nm. High adsorption at 280 nm from 50-450 ml retention time was dueto non-bound protein in the column flow through. The arrow at the peakat 530.09 ml indicates the EpCAM bispecific construct containing proteinfraction that was used or further purification.

FIG. 6:

A representative protein elution pattern from a Sephadex® S200gelfiltration column at 280 nm. The protein peak at 82.66 ml containingbispecific antibodies against CD3 and EpCAM corresponds to a molecularweight of ca. 52 kD. Fractions were collected from 40-140 ml retentiontime.

FIG. 7

A) Cation exchange chromatogram of 3-1 x anti-CD3 (SEQ ID NO.:36) showsthe overall charge isoforms of the protein. Cation exchangechromatography was performed on a MiniS® (Amersham) column. Afterwashing with 20 mM MES buffer pH 5.5, the protein was eluted with agradient of elution buffer containing 1 M NaCl: 0-30% in 60 columnvolumes. The bispecific construct was eluted at 23.58 ml. Unspecificprotein was eluted with 1 M NaCl starting at 50 ml.

B) Cation exchange chromatogram of 5-10 x anti-CD3 (SEQ ID NO.:44) showsthe overall charge isoforms of the protein. Cation exchangechromatography was performed as in FIG. 7A. The bispecific construct waseluted at a shoulder at 35.77 ml. Unspecific protein was eluted with 1 MNaCl starting at 50 ml.

FIG. 8:

A) Representative SDS-PAGE analysis of EpCAM bispecific single chainantibody protein fractions. Lane M: Molecular weight marker Lane 1: cellculture supernatant; lane 2: IMAC flow through; lane 3: IMAC wash; lane4: IMAC eluate; lane 5: purified antibody against EpCAM and CD3 obtainedfrom gel filtration.

B) Representative Western blot analysis of purified EpCAM bispecificsingle chain antibody protein fractions Lane 1: cell culturesupernatant; lane 2: IMAC flow through; lane 3: IMAC wash; lane 4: IMACeluate; lane 5: purified antibody against EpCAM and CD3 obtained fromgel filtration.

FIG. 9:

Cytotoxicity assay of C-terminal EpCAM binders anti-CD3×3-1 (SEQ IDNO.:46), anti-CD3 x-5-10 (SEQ ID NO.:4), and anti-CD3×4-7 (SEQ IDNO.:2). CB15 T cell clone and CHO-EpCAM cells were used in an E:T ratioof 5:1. CHO-EpCAM cells were stained with PKH26 dye and the cells werecounted after bispecific single chain antibody incubation with FACSanalysis.

FIG. 10:

Cytotoxicity assay of N-terminal EpCAM binders 3-1xanti-CD3 (SEQ IDNO.:36), and 5-10xanti-CD3 (SEQ ID NO.:44). CB15 T cell clone andCHO-EpCAM cells were used in an E:T ration of 5:1. CHO-EpCAM cells werestained with PKH26 dye and the cells were counted after bispecificsingle chain antibody incubation with FACS analysis.

FIG. 11:

A) Sequence alignment of the CDR3 of the VH chains of EpCAM 3-1 (SEQ IDNO.: 80), EpCAM 4-1 (SEQ ID NO.: 88), EpCAM 5-10 (SEQ ID NO.: 96), EpCAM3-5 (SEQ ID NO.: 84), EpCAM 4-7 (SEQ ID NO.:92), compared with CDR3 ofthe VH chain of EpCAM M79, HD70 and 3B10. The NXD motif is depicted asbold.

B) Comparison of the cytotoxic activity of 3-1xanti-CD3 (SEQ ID NO.:36), 5-10xanti-CD3 (SEQ ID NO.:44), anti-CD3×4-7 (SEQ ID NO.:2) andanti-CD3×5-10 (SEQ ID NO.:18) with M79Xanti-CD3 and HD70xanti-CD3controls. PBMC cells and Kato III cells were used in a E:T ratio of10:1. KatoIII cells were stained with propidium iodide and the cellswere counted after bispecific single chain antibody incubation with FACSanalysis.

The invention will now be described by reference to the followingbiological examples which are merely illustrative and are not to beconstrued as a limitation of scope of the present invention.

EXAMPLE 1 Cloning and Expression of the EpCAM Constructs

A number of constructs comprising anti-CD3 and anti-EpCAM in variousstructures) and domain arrangements were generated. Anti-EpCAM VH and VLvariable domains of the antibodies 3-1 are shown in SEQ ID NO.:79, 80,81, 82, 3-5 in SEQ ID NO.:83, 84, 85, 86, 4-1 in SEQ ID NO.:87, 88, 89,90, 4-7 SEQ ID NO.:91, 92, 93, 94 and 5-10 in SEQ ID NO.:95, 96, 97, 98.The constructs are summarized in Table 1.

TABLE 1 anti-CD3-anti-EpCAM and anti-EpCAM-anti-CD3 constructs SEQ IDNO.: Domain Distinctive Construct No. Construct arrangement featureanti-CD3xanti-EpCAM constructs SEQ ID NO.: 1, 2 anti-CD3x4-7 VH-VLXVH-VLSEQ ID NO.: 3, 4 anti-CD3x5- VH-VLXVH-VL 10 SEQ ID NO.: 45, 46anti-CD3x3-1 VH-VLXVH-VL SEQ ID NO.: 49, 50 anti-CD3x3-5 VH-VLXVH-VL SEQID NO.: 55, 56 anti-CD3x4-1 VH-VLXVH-VL SEQ ID NO.: 7, 8 anti-CD3xVH-VLXVH-VL Cys-Ser 4-7Cys-Ser mutation SEQ ID NO.: 9, 10 anti-CD3xVH-VLXVH-VL Cys-Ser 5-10Cys-Ser mutation SEQ ID NO.: 47, 48 anti-CD3x3-1VH-VLXVH-VL Cys-Ser mutation SEQ ID NO.: 51, 52 anti-CD3x3-5 VH-VLXVH-VLCys-Ser mutation SEQ ID NO.: 57, 58 anti-CD3x4-1 VH-VLXVH-VL Cys-Sermutation SEQ ID NO.: 11, 12 anti-CD3x3-1 VH-VLXVH-VL (G₄S)₃ -linker SEQID NO.: 13, 14 anti-CD3x4-7 VH-VLXVH-VL (G₄S)₃ -linker SEQ ID NO.: 15,16 anti-CD3x4-7 VH-VLXVL-VH (G₄S)₃ -linker SEQ ID NO.: 17, 18anti-CD3x5- VH-VLXVH-VL (G₄S)₃ -linker 1 10 SEQ ID NO.: 19, 20anti-CD3x5- VH-VLXVL-VH (G₄S)₃ -linker 10 SEQ ID NO.: 53, 54anti-CD3x3-5 VH-VLXVH-VL (G₄S)₃ -linker SEQ ID NO.: 59, anti-CD3x4-1VH-VLXVH-VL (G₄S)₃ -linker 60 anti-EpCAM- anti-CD3 constructs SEQ IDNO.: 29, 30 3-5xanti-CD3 VL-VHxVH-VL SEQ ID NO.: 35, 36 3-1xanti-CD3VL-VHxVH-VL SEQ ID NO.: 38, 39 4-1xanti-CD3 VL-VHxVH-VL SEQ ID NO.: 41,42 4-7xanti-CD3 VL-VHxVH-VL SEQ ID NO.: 43, 44 5-10xanti- VL-VHxVH-VLCD3

1.1 Cloning of C-Terminal EpCAM-Binders 1.1.1 Preparation of Anti-CD3PCR Products

a) Anti-CD3 Constructs with Original 18 Amino Acid Linker (SEQ IDNOs.:1, 2, 3 and 4)

The N-terminal original anti-CD3 containing the 18 amino acid linker(SEQ ID NO.:70) was obtained by PCR using the CD19xCD3 construct(Löffler A et al., Blood 2000 95:2098-103) as template and the followingprimers (CD3 VH BsrGI: AGGTGTACACTCCGATATCAAACTGCAGCAG (SEQ ID NO.:5),CD3 VL BspEI: AATCCGGATTTCAGCTCCAGCTTGG (SEQ ID NO.:6)).

b) Anti-CD3 Constructs with Original 18 Amino Acid Linker and Cys to SerMutation in CDRH3 (SEQ ID Nos. 7,8, 9 and 10)

The N-terminal original anti-CD3 containing the 18 amino acid linker(Seq ID NO.:70) and the Cys to Ser mutation was obtained by PCR using aCD19xanti-CD3 (C→S mutation) construct as template and the primers CD3VH BsrGI and CD3 VL BspEI (Seq ID Nos. 5 and 6). The CDRH3 sequence withthe Cys-Ser mutation is shown in SEQ ID NO.:78.

c) Anti-CD3-Anti-EpCAM Constructs with (G4S)3 Linker (Seq ID Nos. 11,12, 13, 14, 15, 16, 17, 18, 19 and 20)

The N-terminal anti-CD3 containing the 15 amino acid standard (G₄S)₃linker, (SEQ ID NO.:99) was obtained by PCR using the CD19xCD3 (LöfflerA et al., Blood 2000 95:2098-103) as template. The anti-CD3 VH regionand the anti-CD3 VL region were separately amplified by the followingprimers (CD3 VH: CD3 VH BsrG1 AGGTGTACACTCCGATATCAAACTGCAGCAG (SEQ IDNO.:5), 3″CD3 VH GS15GGAGCCGCCGCCGCCAGAACCACCACCACCTGAGGAGACTGTGAGAGTGGTGCCTTG (SEQ IDNO.:21); CD3 VL: 5″CD3 VL GS15GGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATTCAGCTGACCCAGTCTCC (SEQ ID NO.:22),CD3 VL BspEI AATCCGGATTTCAGCTCCAGCTTGG (SEQ ID NO.:6)). Overlappingcomplementary sequences introduced into the PCR products were used toform the coding sequence of a 15-amino acid (G₄S)₃ (single-letter aminoacid code) (SEQ ID NO.:99) linker during the subsequent fusion PCR. Thisamplification step was performed with the primer pair CD3 VH BsrGI (SEQID NO.:5) and CD3 VL BspEI (SEQ ID NO.:6).

1.1.2 Cloning of the Anti-CD3xanti EpCAM Constructs inVH_(anti-CD3)-VL_(anti-CD3) x VH_(anti-EpCAM)-VL_(anti-EpCAM)Orientation (SEQ ID NO.:1,2, SEQ ID NO.:3,4, SEQ ID NO.:7,8, SEQ IDNO.:9,10, SEQ ID NO.:11,12, SEQ ID NO.:13,14 and SEQ ID NO.:17,18)

The N-terminal original anti-CD3 containing the 18 amino acid linker(SEQ ID NO.:70) or the N-terminal original anti-CD3 containing the 15amino acid standard (G₄S)₃ linker (SEQ ID NO.:99) was cleaved with therestriction enzymes BsrGI and BspEI and subsequently cloned into thebluescript KS vector (Stratagene, La Jolla, Calif.), containing theamino acid sequence of an eukaryotic secretory signal (leader peptide)as a EcoRI/BsrGI-Fragment. After cleavage of this construct with EcoRIand BspEI the resulting DNA fragment comprising the respective anti-CD3scFv with the leader peptide was cloned into a EcoRI/BspEI cleavedplasmid containing the c-terminal EpCAM binders 3-1 (SEQ ID NO.:79-82),4-7 (SEQ ID NO.:91-94), or 5-10 (SEQ ID NO.:95-98) in pEFDHFR. pEFDHFRwas described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995)7021-7025).

1.1.3. Cloning of the Anti-CD3xanti EpCAM Constructs inVH_(anti-CD3)-VL_(anti-CD3) x VL_(anti-EpCAM)-VH_(anti-EpCAM)Orientation (SEQ ID Nos.: 15, 16, 19 and 20)

The C-terminal anti-EpCAM antibody 4-7 (SEQ ID NO.:91-94) in VLVHorientation containing the 15 amino acid standard linker (SEQ ID NO.:99)was obtained by PCR. The 4-7 VH region and the 4-7 VL region wereseparately amplified by the following primers (4-7 VL: 4-7. VL BspEI FORCTGAAATCCGGAGGTGGTGGATCCGAGCTCGTGATGACCCAGACTCC (SEQ ID NO.:100), 4-7 VLGS15 REV GGAGCCGCCGCCGCCAGAACCACCACCACCTTTGATCTCAAGCTTGGTCCCC (SEQ IDNO.:101); 4-7 VH: 4-7 VH GS15 FORGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGGTGCAGCTGCTCGAGCAG (SEQ ID NO.:23), 4-7VH SalI REV TTTTAAGTCGACCTAATGATGATGATGATGATGTGAGGAGACGGTGACCGTGG (SEQID NO.:24)). Overlapping complementary sequences introduced into the PCRproducts were used to form the coding sequence of a 15-amino acid (G₄S)₃(single-letter amino acid code) linker (SEQ ID NO.:99) during thesubsequent fusion PCR. This amplification step was performed with theprimer pair 4-7 VL BspEI FOR and 4-7 VH SalI REV (SEQ ID NO.100, SEQ IDNO.:24).

The C-terminal anti-EpCAM antibody 5-10 (SEQ ID NO.:95-98) in VLVHorientation containing the 15 amino acid standard linker (SEQ ID NO.:99)was obtained by PCR. The 5-10 VH region and the 5-10 VL region wereseparately amplified by the following primers (5-10 VL: 5-10 VL BspEIFOR CTGAAATCCGGAGGTGGTGGATCCGAGCTCGTGATGACACAGTCTCCAT (SEQ ID NO.:25),5-10 VL GS15 REV GGAGCCGCCGCCGCCAGAACCACCACCACCTTTGATCTCAAGCTTGGTCCCAG(SEQ ID NO.: 26); 5-10 VH: 5-10 VH GS15 FORGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGGTGCAGCTGCTCGAGC (SEQ ID NO.:27), 5-10VH SalI REV TTTTAAGTCGACCTAATGATGATGATGATGATGTGAGGAGACGGTGACCGTGG (SEQID NO.:28)). Overlapping complementary sequences introduced into the PCRproducts were used to form the coding sequence of a 15-amino acid (G₄S)₃linker (SEQ ID NO.:99) during the subsequent fusion PCR. Thisamplification step was performed with the primer pair 5-10 VL BspEI FORand 5-10 VH SalI REV (SEQ ID NO.:25, SEQ ID NO:28).

These PCR products (5-10 VLVH and 4-7 VLVH) were cleaved with BspEI andSa/I and ligated in the BspEI/SalI cleaved anti-CD3 VHVL stLx5-10 VHVL(SEQ ID NO.:17,18) or anti-CD3 VHVL stL x 4-7 (SEQ ID NO.:13, 14) VHVLin pEFDHFR replacing the 5-10 VHVL DNA fragment.

1.1.4. Expression and Binding of the Anti-CD3-EpCAM Constructs

After confirmation of the sequence coding for the bispecific singlechain by sequencing the plasmid was transfected into DHFR deficient CHOcells for eukaryotic expression. Eukaryotic protein expression in DHFRdeficient CHO cells was performed as described in Kaufmann R. J. (1990)Methods Enzymol. 185, 537-566). The transfected cells were then expandedand 1 litre of supernatant produced. Expression and binding of thebispecific single chain molecules were confirmed by FACS analyses. Forthat purpose the EpCAM positive human gastric cancer cell line Kato III(obtained from American Type Culture Collection (ATCC) Manassas, Va.20108 USA, ATCC number: HTB-103) was used. Binding of the anti-CD3 partwas demonstrated on Jurkat cells (ATCC TIB 152).

Cells were cultured according to the recommendations of the supplier andca. 200000 cells were incubated with 10 μg/ml of the construct in 50 μlPBS with 2% FCS. The binding of the construct was detected with ananti-His antibody (Penta-His Antibody, BSA free, obtained from QuiagenGmbH, Hilden, FRG) at 2 μg/ml in 50 μl PBS with 2% FCS. As a second stepreagent a R-Phycoerythrin-conjugated affinity purified F(ab′)₂ fragment,goat anti-mouse IgG, Fc-gamma fragment specific antibody, diluted 1:100in 50 μl PBS with 2% FCS (obtained from Dianova, Hamburg, FRG) was used.The samples were measured on a FACSscan (BD biosciences, Heidelberg,FRG). All the constructs comprising anti-CD3 and anti-EpCAM showedstronger binding affinity to CD3 and to EpCAM than the prior artanti-EpCAM (M79)xanti-CD3 bispecific antibody (FIG. 2).

1.2 N-Terminal EpCAM Binders 1.2.1 Cloning of the Anti-EpCAMxanti-CD3Constructs

Cloning of the Construct 3-5xanti-CD3 (SEQ ID NOs.29, 30):

The C-terminal 3-5 in VH-VL orientation was obtained by PCR for theconstruction of 3-5 xanti-CD3 (SEQ ID NO.:29) molecule. Fragments I andII were amplified by PCR using primer pairs me 81 (SEQ ID NO.:31)/me 90(SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ ID NO.:33),respectively. Hot Start PCR was done using the Expand High FidelitySystem of Roche Diagnostics. 20 cycles (94° C./30 sec; 60° C./1 min; 72°C./1 min) were used for amplification followed by one cycle of 3 min at72° C.

PCR fragments I and II were subjected to electrophoresis on a 1.5%agarose gel. Fragments were mixed (1 ng of each) and used as a templatefor the next PCR reaction performed with primer pair me 81 (SEQ IDNO.:31) and me 84 (SEQ ID NO:33) for amplification of fragment III. PCRwas performed as described above. Fragment III was purified on anagarose gel and digested with BssHII and BspEI (Biolabs), purified andsubsequently cloned into the corresponding sites of the pEF-dHFR-signalpeptide (77/78)-anti-CD3 cloning vector, which facilitates cloning ofanti-target variable regions in front of the anti-CD3 region. The vectorhas a unique BssHII site just after the signal peptide followed by BspEIsite, linker (G₄S) and anti-CD3 region. The cloned region was verifiedby restriction digests and by DNA-sequencing.

Sequences of the Primers Used:

Me 81: (SEQ ID NO.: 31) 5′-GGA TGC GCG CGA GCT CGT GAT GAC CCA GAC TCCACTC TCC-3′ Me 83: (SEQ ID NO.: 32)5′-GGT TCT GGC GGC GGC GGC TCC GGT GGT GGT GGTTCT GAG GTG CAG CTG CTC GA CAG TCT G-3′ Me 84: (SEQ ID NO.: 33)5′-GTG CTC CGG AGG AGA CGG TGA CCG TGG TCC CTT GGC CCC AG-3′ Me 90:(SEQ ID NO.: 34) 5′-CCG GAG CCG CCG CCG CCA GAA CCA CCA CCA CCTTTG ATC TCA AGC TTG GTC CC-3′Cloning of the Construct 3-1xanti-CD3 (SEQ ID NO.:35, 36),

The C-terminal 3-1 in VH-VL orientation was obtained by PCR for theconstruction of 3-1 xanti-CD3 (SEQ ID NO.:35) molecule. Fragments I andII were amplified by PCR using primer pairs me 91a (SEQ ID NO.:37)/me 90(SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ ID NO.:33),respectively. PCR was performed as above.

Agarose gel fragments comprising PCR fragments I and II were used as atemplate for the next PCR reaction performed with primer pair me 91a(SEQ ID NO.:37) and me 84 (SEQ ID NO.:33) for amplification of fragmentIII. PCR was performed as described above except, annealing wasperformed at 68° C. instead of at 60° C. Fragment III was purified on anagarose gel and digested with BsrGI and BspEI (Biolabs), purified andsubsequently cloned into the corresponding sites of the pEF-dHFR-M79 Xanti-CD3 cloning vector. The cloned region was verified by restrictiondigests and by DNA-sequencing.

Me 91a: (SEQ ID NO.: 37)5′-GGA TTG TAC A CTCC GA GCT CGT CAT GAC CCA GTCTCC ATC TTA TCT TGC TGC-3′Cloning of the Construct 4-1 xanti-CD3 (SEQ ID NO.:38, 39):

The C-terminal 4-1 in VH-VL orientation was obtained by PCR for theconstruction of 4-1 xanti-CD3 (SEQ ID NO.:38, 39) molecule. Fragments Iand II were amplified by PCR using primer pairs me 92a (SEQ IDNO.:40)/me 90 (SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ IDNO.:33), respectively. PCR was performed as above in annealingtemperature of 60° C.

Agarose gel fragments comprising PCR fragments I and II were used as atemplate for the next PCR reaction performed with primer pair me 92a(SEQ ID NO.:40) and me 84 (SEQ ID NO.:33) for amplification of fragmentIII. PCR was performed as described above except, annealing wasperformed at 68° C. instead of at 60° C. Fragment III was purified on anagarose gel and digested with BsrGI and BspEI (Biolabs), purified andsubsequently cloned into the corresponding sites of the pEF-dHFR-M79 Xanti-CD3 is cloning vector. The cloned region was verified byrestriction digests and by DNA-sequencing.

Me 92a: (SEQ ID NO.: 40) 5′-GGA TTG TAC A CTCC GA GCT CGT GAT GAC ACAGTCTCC ATC CTC C-3′Cloning of the Construct 4-7xanti-CD3 (SEQ ID NO.:41,42)

The C-terminal 4-7 in VH-VL orientation was obtained by PCR for theconstruction of 4-7 xanti-CD3 (SEQ ID NO.:41, 42) molecule. Fragments Iand II were amplified by PCR using primer pairs me 81 (SEQ ID NO.:31)/me90 (SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 SEQ ID NO.:33),respectively. PCR was performed as above with an annealing temperatureof 60° C.

Agarose gel fragments comprising PCR fragments I and II were used as atemplate for the next PCR reaction performed with primer pair me 81 (SEQID NO.:31) and me 84 (SEQ ID NO.:33) for amplification of fragment III.PCR was performed as described above. Fragment III was purified on anagarose gel and digested with BssHII and BspEI (Biolabs), purified andsubsequently cloned into the corresponding sites of the pEF-dhfr-signalpeptide (77/78)-anti-CD3 cloning vector. The cloned region was verifiedby restriction digests and by DNA-sequencing.

Cloning of the Construct 5-10xanti-CD3 (SEQ ID NO.:43, 44)

The C-terminal 5-10 in VH-VL orientation was obtained by PCR for theconstruction of 5-10xanti-CD3 (SEQ ID NO.:43, 44) molecule. Fragments Iand II were amplified by PCR using primer pairs me 92a (SEQ IDNO.:40)/me 90 (SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ IDNO.:33), respectively. PCR was performed as above with an annealingtemperature of 60° C.

Agarose gel fragments comprising PCR fragments I and II were used as atemplate for PCR with primer pair me 92a (SEQ ID NO.:40) and me 84 (SEQID NO.:33) for amplification of fragment III. PCR was performed asdescribed above except, annealing was performed at 68° C. instead of at60° C. Fragment III was purified on an agarose gel and digested withBsrGI and BspEI (Biolabs), purified and subsequently cloned into thecorresponding sites of the pEF-dhfr-M79 X anti-CD3 cloning vector. Thecloned region was verified by restriction digests and by DNA-sequencing.

1.2.2 Expression of Anti-EpCAMxanti-CD3 Bispecific Molecules

CHO-cells lacking DHFR gene were maintained in alpha MEM medium (LifeTechnologies, cat. no: 32561) supplemented with 10% fetal Calf Serum(Life Technologies, heat inactivated at 65° C. for 30 minutes) and withHT (Hypoxanthin and Thymidine; Life Technologies, cat. no: 41065-012).The cells were transfected with pEF-dHFR-3-1xanti-CD3 (SEQ ID NO.:35,36), pEF-dHFR-3-5xanti-CD3 (SEQ ID NO.:29, 30), pEF-dHFR-4-1xanti-CD3(SEQ ID NO.:38, 39), pEF-dHFR-4-7xanti-CD3 (SEQ ID NO.:41, 42) andpEF-dHFR-5-10xanti-CD3 (SEQ ID NO.:43, 44) using Lipofectamine 2000 kit(Invitrogen; cat. no:11668-019) according to the instructions providedby the Manufacturer. After 48 hrs, the cells were subjected to selectionby transferring the transfected cells into the selection medium (alphaMEM medium (cat. no:32561) containing heat inactivated 10% dialysedfetal Calf Serum (Life Technologies). After 2-3 weeks of selection, thecells were grown for 8 to 9 days (in 500 ml of selection medium) forproduction of bispecific molecules in 2 litre Tissue culture RollerBottles (Falcon (cat. no: 353068; Becton Dickinson Labware). The tissueculture medium was centrifuged at 4° C. for 10 minutes at 300 g (1300rpm) to remove the cells and cell debris. The supernatant containing thesecreated bispecific molecules was stored at −20° C. until furtheranalysis.

1.2.3 Binding Assays of Bispecific Anti EpCAMxanti CD3 Variants

In order to analyze the binding strength of the bispecificanti-EpCAMxanti-CD3 single chain constructs of the invention, thefollowing binding assay was carried out.

250000 Jurkat cells (for CD3 binding) and Kato cells (for EpCAM binding)were independently incubated with crude supernatants (50 μl) containingbispecific construct for 45 min. at 4° C. Thereafter, the cells werewashed twice in FACS buffer (phosphate-buffered saline containing 1%fetal calf serum (FCS) and 0.05% sodium azide) and incubated with mouseanti-His antibody (Dianova, DIA910) for 60 min. at 4° C. Washing stepswere performed as above.

The cells were finally incubated either with goatanti-mouse-FITC-conjugated antibody (BD 550003) or with anti-mouse-PEconjugated antibody (IgG) (Sigma, P8547). After washing steps, 10,000events were analysed using FACS Calibur (B&D). All the EpCAM constructsshowed strong binding (FIG. 4).

EXAMPLE 2 Purification of the EpCAM Constructs

In order to purify the bispecific single chain constructs comprisinganti-EpCAM and anti-CD3 the CHO-EpCAM cells were grown in roller bottleswith HiClone® CHO modified DMEM medium (HiQ) for 7 days before harvest.The cells were removed by centrifugation and the supernatant containingthe expressed protein was stored at −20° C.

Äkta FPLC System® (Pharmacia) and Unicorn Software® were used forchromatography. All chemicals were of research grade and purchased fromSigma (Deisenhofen) or Merck (Darmstadt).

IMAC was performed, using a Fractogel® column (Pharmacia) that wasloaded with ZnCl₂ according to the manufacturers protocol. The columnwas equilibrated with buffer A2 (20 mM NaPP pH 7.5, 0.4 M NaCl) and thecell culture supernatant (500 ml) was applied to the column (10 ml) witha flow rate of 3 ml/min. The column was washed with buffer A2 to removeunbound sample. Bound protein was eluted using a 2-step gradient ofbuffer B2 (20 mM NaPP pH 7.5, 0.4 M NaCl, 0.5 M Imidazol). In Step 1 20%buffer B2 in 10 column volumes was used and in Step 2 100% buffer B2 in10 column volumes was used. Eluted protein fractions from the 100% stepwere pooled for further purification.

Gel filtration chromatography was performed on a Sephadex S200 HiPrep®column (Pharmacia) equilibrated with PBS (Gibco). Eluted protein samples(flow rate 1 ml/min) were subjected to SDS-Page and Western Blot fordetection.

The column was previously calibrated for molecular weight determination(molecular weight marker kit, Sigma MW GF-200).

Protein concentrations were determined using protein assay dye(MicroBCA, Pierce) and IgG (Biorad) as standard protein. The yields ofthe protein are shown in Table 2. All constructs were producible.

TABLE 2 Yields of the single-chain bispecific constructs comprisinganti-EpCAM and anti-CD3 Yield [μg purified protein per Construct literculture] 4-1 x anti-CD3 (SEQ ID NO.: 39) 172.5 3-5 x anti-CD3 (SEQ IDNO.: 30) 265 4-7 x anti-CD3 (SEQ ID NO.: 42) 37 anti-CD3 x 4-7. (SEQ IDNO.: 2) 112.5 anti-CD3 Cys-Ser x 4-7 (SEQ ID 140 NO.: 8) 3-1 x anti-CD3(SEQ ID NO.: 36) 265 5-10 x anti-CD3 (SEQ ID NO.: 44) 400 anti-CD3 x5-10 (SEQ ID NO.: 4) 195

A further high resolution cation exchange chromatography was performedon a MiniS® column (Amersham), equilibrated with 20 mM MES buffer pH5.5. The sample was diluted 1:3 with the same buffer before loading tothe column. Bound protein was eluted with a gradient of equilibrationbuffer containing 1M NaCl: 0-30% in 60 column volumes. Remaining proteinwas eluted in 3 column volumes of 1M NaCl (FIG. 7).

The EpCAM bispecific single chain construct proteins were isolated in atwo-step purification process including immobilized metal affinitychromatography (IMAC) (FIG. 5) and gel filtration (FIG. 6). The mainproduct had a molecular weight of 52 kDa under native conditions asdetermined by gelfiltration in PBS.

Purified bispecific protein was analyzed in SDS PAGE under reducingconditions performed with precast 4-12% Bis Tris gels (Invitrogen).Sample preparation and application were according to the manufacturersprotocol. The molecular weight was determined with MultiMark® proteinstandard (Invitrogen). The gel was stained with colloidal Coomassie(Invitrogen protocol). The purity of the isolated protein was shown tobe >95% (FIG. 8A). Western Blot was performed with an Optitran BA-S83®membrane and the Invitrogen Blot Module according to the manufacturersprotocol. The antibodies used were Penta H is (Qiagen) andGoat-anti-Mouse-Ig labeled with alkaline phosphatase (AP) (Sigma), thechromogenic substrate solution was BCIP/NBT liquid (Sigma). The EpCAMbispecific protein could be specifically detected by Western Blot (FIG.8B). The main signal corresponds to the main band in the SDS PAGE at 52kD corresponding to the purified bispecific molecule.

EXAMPLE 3 Cytotoxicity Assays of the Constructs Comprising Anti-CD3 andAnti-EpCAM

In order to test the bioactivity of the constructs comprising anti-EpCAMand anti-CD3 a FACS based cytotoxicity test was performed.

For the cytotoxicity test, CHO cells from the American Type Cell CultureCollection (ATCC, Manassas, USA) were transfected with epithelial celladhesion molecule (EpCAM). A cell clone derived from this transfection,referred to as CHO-EpCAM cells, was used for the experiments. CHO-EpCAM(1.5×10⁷) cells were washed free of serum two times with PBS andincubated with PKH26 dye (Sigma-Aldrich Co.) according to themanufacturers instructions. After staining cells were washed two timeswith RPMI/10% FCS.

Cells were counted and mixed with CB15 effector cells. The CD4-positiveT cell clone CB15 was provided by Dr. Fickenscher, University ofErlangen/Nuernberg, Germany. Cells were cultured as recommended by thesuppliers. The resulting cell suspension contained 400.000 target and2×10⁶ effector cells per ml. 50 μl of the mixture was used per well in a96 well round bottom plate.

Antibodies were diluted in RPMI/10% FCS to the required concentrationand 50 μl of this solution was added to the cell suspension. A standardreaction was incubated for 16 h at 37° C./5% CO₂. Propidium iodide wasadded to a final concentration of 1 μg/ml. After 10 min of incubation atroom temperature cells were analysed by FACS. PKH26 fluorescence wasused for positive identification of target cells. Cytotoxicity wasmeasured as ratio of PI positive over all target cells. Sigmoidal doseresponse curves typically had R² values >0.97 as determined by PrismSoftware (GraphPad Software Inc., San Diego, USA) (FIGS. 9 and 10). EC₅₀values calculated by the analysis program were used for comparison ofbioactivity. All the constructs of the invention show at least 50 timesbetter cytotoxicity (maximum EC50-value 169 pg/ml) than the prior artconstruct M79xanti-CD3 (8628 pg/ml).

EXAMPLE 4 Determination of the Binding Affinity by BIAcore™ 2000 of theConstructs Comprising Anti-EpCAM and Anti-CD3 to EpCAM

In order to show the superior binding affinity of the constructs of theinvention, the KD values of the constructs and of the prior artanti-EpCAM construct (M79)xanti-CD3 were determined.

Kinetic binding experiments were performed using surface plasmonresonance on the BIAcore™ 2000, Biacore AB (Uppsala, Sweden) with a flowrate of 5 μL/min and HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mMEDTA, 0.005% surfactant P20) as running buffer at 25° C. Theextracellular domain of the EpCAM antigen (residues 17-265) wasimmobilized onto flow cells 2-4 on a CM5 sensor chip. The chip surfacewas activated injecting 80 μL of 0.1 M sodium-hydroxysuccinimid, 0.4 MN-ethyl-N′(3-dimethylaminepropyl)-carbodiimid (NHS/EDC). The antigen wascoupled by manual injection of 60 μg/mL EpCAM in 0.01 M sodium-acetate,pH 4.7. Different densities of antigen were immobilized on flow cells2-4 adjusting the amount of manual injection times. Flow cell 1 was leftempty while flow cell 2 was coated with the highest density of EpCAM(4100 RU). Flow cell 3 was coated with ¼ of the amount of antigenimmobilized on flow cell 2 (974 RU) and flow cell 4 was coated withlowest density of Ep-CAM antigen (265 RU). The activated surface of thesensor chip was blocked injecting 85 μL of 1 M ethanolamine and the chipwas left to equilibrate over night at a constant flow of 5 μL/min ofHBS-EP.

Binding kinetics of the bispecific constructs were measured injecting 10μL of protein solution at concentrations ranging from 4 μM-0.07 μM andmonitoring the dissociation for 100 sec. Protein was buffered in HBS-EP.The data were fitted using BIAevalution™ software determining the rateconstant for dissociation and association kinetics with a 1:1 Langmuirbinding equation (1, 2). Where A is the concentration of injectedanalyte and B[0] is Rmax.

dB/dt=−(ka*[A]*[B]−kd*[AB])  (1)

dAB/dt=−(ka*[A]*[B]−kd*[AB])  (2)

Kinetic binding curves were determined in four concentrations of eachbispecific construct analysed. The independent fitting of the raw dataresulted in dissociation and association rate constants that were usedto calculate the equilibrium dissociation constant (KD). The calculatedKD values were unbiased for concentration indicating reliable dataanalysis. The average of the independently determined dissociationconstants as well as the standard deviation are summarized in table 3.

The analysed bispecific constructs bind to the Ep-CAM antigenimmobilized on the chip surface within a well defined affinity range.The standard deviation for the calculated average dissociation constantis as expected.

TABLE 3 Dissociation constants for the bispecific constructs binding toEpCAM. KD (M) M79 x anti-CD3 (control) 4.0 × 10⁻⁶ 4-1 x anti-CD3 (SEQ ID2.5 × 10⁻⁷ NO.: 39) 3-5 x anti-CD3 (SEQ ID 2.3 × 10⁻⁷ NO.: 30)

The prior art anti-EpCAM x anti-CD3 construct M79xCD3 had a KD of4.0×10⁻⁶ M while surprisingly the constructs of the invention have a KDin the range of 2.3×10⁻⁷-2.5×10⁻⁷ M. Thus, the constructs of theinvention have more than 15 times stronger binding affinity than theprior art construct.

EXAMPLE 5 Comparison of the Cytotoxic Activity of the Constructs of theInvention with Prior Art Constructs

In order to compare the bioactivity of constructs having the NXD motifwith conventional M79xCD3 and HD70xCD3 constructs the followingcytotoxic assay was carried out.

KatoIII cells (ATCC HTB-103) were used as target cells and grown in RPMIsupplemented with 10% fetal calf serum at 37° C. in a 5% CO₂ humidifiedincubator. Subconfluent cultures were treated with 0.25% trypsin,counted in a Neubauer chamber slide and checked for vitality bytrypan-blue exclusion. Only cultures with >95% vitality were used forcytotoxicity assays. Target cells were stained with PKH26 fluorescentmembrane dye according to the manufacturers manual (Sigma-Aldrich GmbH,Germany, PKH26-GL). Cell number was adjusted to 8×10⁵ cells/ml in RPMIcomplete medium.

Human peripheral blood mononuclear cells (PBMCs) were used as effectorcells and isolated from healthy donors using ficoll density gradientcentrifugation with subsequent 100×g centrifugation to removethrombocytes. The pellet was resuspended in 10 vol. erythrocyte lysingbuffer and incubated at room temperature for 10 min. Lysing reaction wasstopped by addition PBS. PBMCs were resuspended in RPMI 1640 completemedium and cell number adjusted to 8×10⁶ cells/ml.

Equal volumes of target and effector cell suspension were mixed and 50μl of this suspension transferred to each well of a 96 well round bottomplate, 50 μl of EpCAM bispecific antibody serial dilution or RPMIcomplete medium as a negative control was added. Plates were incubatedfor 16 to 20 hrs at 37° C., 5% CO₂ in a humidified incubator. 50 μlpropidium iodide was added to a final concentration of 1 μg/ml andincubated 15 min at room temperature. Samples were analysed by flowcytometry (FACSCalibur, Becton Dickinson). 2×10⁴ events were counted.

Target cells were identified by their PKH26 fluorescent label andcytotoxicity within this population was subsequently determined. Viablecells were separated from dead cells by propidium iodide staining andthe percentage of dead target cells was used as a measure forcytotoxicity. Mean values were plotted against the concentration of thebispecific antibody on a logarithmic scale, resulting in a dose responsecurve (FIG. 11B). The corresponding EC₅₀ values were obtained afternonlinear fitting of data with the GraphPad Prism software.

The cytotoxic activity of constructs having the NXD motif (SEQ IDNO.:36, 44, 2 and 18) was compared with conventional constructsM79xanti-CD3 and HD70-xanti-CD3 (FIG. 11B). A sequence alignment of theCDR3 regions of the VH chains of 3-1, 5-10, 4-7, 3-5 and 4-1 with M79,HD70 and 3B10 is shown in FIG. 11A. Only 3-1, 5-10, 4-7, 3-5 and 4-1have the NXD motif and furthermore, the lengths of the CDR3 regionsdiffer. As can be seen from FIG. 11A, 3-1, 4-1 and 5-10 have a CDR-H3region of 10 amino acids, 3-5 and 4-7 have 14 amino acids whereas theprior art M79 has 8 amino acids, 3B10 has 6 amino acids and HD70 has 18amino acids.

SEQ ID NO.: 36, 44, 2 and 18 showed a clearly better bioactivitycompared to the conventional M79 and HD70 constructs (2250 pg/ml andless compared to 71460 and 11327 pg/ml of the prior art constructs,respectively) demonstrating the advantageous effects of the constructsof the invention.

1. A bispecific single chain antibody construct, whereby said constructcomprises at least two domains, whereby one of said domains binds tohuman EpCAM antigen and a second domain binds to human CD3 antigen,wherein said binding domain specific for EpCAM comprises at least oneCDR-H3 region comprising the amino acid sequence NXD preferably inposition 102 to 104 of SEQ ID NOs: 80,88 and 96, or preferably inposition 106 to 108 of SEQ ID NOs: 84 and 92, wherein X is an aromaticamino acid.
 2. The bispecific single chain antibody construct of claim1, wherein X is W or Y.
 3. The bispecific single chain antibodyconstruct of claim 1, wherein the CDR-H3 comprises at least 9 amino acidresidues.
 4. The bispecific single chain antibody construct of claim 1,wherein said binding domain specific for EpCAM has a K_(D) value of morethan 5×10⁻⁹ M.
 5. The bispecific single chain antibody construct ofclaim 1, wherein said binding domain specific for EpCAM has a K_(D)value in a range between 1×10⁻⁷ and 5×10⁻⁹ M and said binding domainspecific for CD3 has a K_(D) value in a range between 1×10⁻⁶ and 5×10⁻⁹M.
 6. A pharmaceutical composition comprising a bispecific single chainantibody construct, whereby said construct comprises at least twodomains, whereby one of said at least two domains specifically binds tohuman EpCAM antigen and a second domain binds to human CD3 antigen,wherein said binding domain specific for EpCAM comprises at least oneCDR-H3 region of at least 9 amino acid residues and wherein said bindingdomain specific for EpCAM has a K_(D) value of more than 5×10⁻⁹ M. 7.The bispecific single chain antibody construct of claim 1, wherein saidbinding domain specific for CD3 has a K_(D) value of more than 10⁻⁷ M.8. The bispecific single chain antibody construct of claim 1, whereinthe CDR-H3 region comprises at least 14 amino acids.
 9. The bispecificsingle chain antibody construct of claim 1, wherein the V_(H) chaindomains specific for human EpCAM antigen is selected from the groupconsisting of: (a) an amino acid sequence as shown in any of SEQ ID NO:80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92 and SEQ ID NO: 96; (b)an amino acid sequence encoded by a nucleic acid sequence as shown inSEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91 and SEQ IDNO: 95; (c) an amino acid sequence encoded by a nucleic acid sequencehybridizing with the complementary strand of a nucleic acid sequence asdefined in (b) under stringent hybridization conditions; and (d) anamino acid sequence encoded by a nucleic acid sequence which isdegenerate as a result of the genetic code to a nucleotide sequence ofany one of (b) and (c).
 10. The bispecific single chain antibodyconstruct of claim 1, wherein the V_(L) chain domains specific for humanEpCAM antigen is selected from the group consisting of: (a) an aminoacid sequence as shown in any of SEQ ID NO: 82, SEQ ID NO: 86, SEQ IDNO: 90, SEQ ID NO: 94 and SEQ ID NO: 98; (b) an amino acid sequenceencoded by a nucleic acid sequence as shown in SEQ ID NO: 81, SEQ ID NO:85, SEQ ID NO: 89, SEQ ID NO: 93 and SEQ ID NO: 97; (c) an amino acidsequence encoded by a nucleic acid sequence hybridizing with thecomplementary strand of a nucleic acid sequence as defined in (b) understringent hybridization conditions; and (d) an amino acid sequenceencoded by a nucleic acid sequence which is degenerate as a result ofthe genetic code to a nucleotide sequence of any one of (b) and (c). 11.The bispecific single chain antibody construct of claim 1, wherein thebinding domains specific for the CD3 antigen is derived from an antibodyselected from the group consisting of: X35-3, VIT3, BMA030 (BW264/56),CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4. 2, TR-66, WT32, SPv-T3b,11 D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D,M-T301, SMC2, WT31 and F101.01.
 12. The bispecific single chain antibodyconstruct of claim 1, wherein said bispecific single chain antibodyconstruct comprises an amino acid sequence selected from the groupconsisting of (a) an amino acid sequence as shown in any of SEQ ID NOs:2, 4, 8, 10, 12, 14, 16, 18, 20, 30, 36, 39, 42, 44, 46, 48, 50, 52, 54,56, 58 and 60; (b) an amino acid sequence encoded by a nucleic acidsequence as shown in any of SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15, 17, 19,29, 35, 38, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59; (c) an amino acidsequence encoded by a nucleic acid sequence hybridizing with thecomplementary strand of a nucleic acid sequence as defined in (b) understringent hybridization conditions; and (d) an amino acid sequenceencoded by a nucleic acid sequence which is degenerate as a result ofthe genetic code to a nucleotide sequence of any one of (b) and (c). 13.An isolated nucleic acid sequence encoding a bispecific single chainantibody construct of claim
 1. 14. A vector which comprises a nucleicacid sequence of claim
 13. 15. The vector of claim 14, wherein saidvector further comprises a regulatory sequence which is operably linkedto said nucleic acid sequence of claim
 13. 16. The vector of claim 14,wherein said vector is an expression vector.
 17. A host transformed ortransfected with a vector of claim
 14. 18. A pharmaceutical compositionof claim 26, further comprising a proteinaceous compound capable ofproviding an activation signal for immune effector cells.
 19. Thepharmaceutical composition of claim 18, wherein the pharmaceuticalcomposition is thermostable at ≧37° C.
 20. A process for the productionof a bispecific single chain antibody construct, said process comprisingculturing the host of claim 17 under conditions allowing the expressionof the bispecific single chain antibody construct and recovering theproduced bispecific single chain antibody construct from the culture.21. (canceled)
 22. A method for the prevention, treatment oramelioration of a tumorous disease, comprising the step of administeringto a subject in need of such a prevention, treatment or amelioration apharmaceutical composition of claim
 26. 23. The method of claim 22,wherein said subject is a human.
 24. The method of claim 22, whereinsaid tumorous disease is epithelial cancer or a minimal residual cancer.25. A kit comprising a bispecific single chain antibody construct ofclaim
 1. 26. A pharmaceutical composition comprising the bispecificsingle chain antibody construct of claim 1.